A 6-month histological analysis on maxillary sinus augmentation with and without use of collagen membranes over the osteotomy window: randomized clinical trial.

INTRODUCTION: Over the years, several modifications have been made to the sinus augmentation technique and to the materials used. However, there is still controversy about the need for using a barrier concurrently with a graft in sinus augmentation procedures. On this basis, the aim of this randomized clinical study was to investigate the effect of resorbable collagen membrane over the osteotomy window on maxillary sinus augmentation healing. MATERIALS AND METHODS: Patients who required maxillary sinus augmentation were evaluated and selected to enter the study. After maxillary sinus grafting, each patient was randomly assigned to control (membrane over the osteotomy window) or test (no membrane) group. After 6 months, one bone biopsy was harvested from the lateral window and sent to the histology laboratory. The Mann-Whitney nonparametric test was used for comparing the two groups. P-value was set at 5%. RESULTS: Eighteen patients entered the study and were randomly allocated in control (nine patients) or test group (nine patients). The histomorphometric measurements revealed that newly formed bone was 30.7% ± 15.5% of the total volume from the membrane group (control). The average percentage of connective tissue was 50.6% ± 18.7% and residual graft percentage was 18.4% ± 20.3%. On the other hand, data regarding the nonmembrane group (test) showed that the percentage of newly formed bone was 28.1% ± 19.4%. The mean percentage of connective tissues was 59.3% ± 15.4% and 12.6% ± 12.4% for the residual graft particles. No significant difference was detected in the histomorphometrical evaluation between the two groups. DISCUSSION: Our results showed that, compared with sites not covered, the use of the membrane did not substantially increase the amount of vital bone over a period of 6 months. On the other hand, the use of membrane seems to reduce the proliferation of the connective tissue and the graft re-absorption rate. It is plausible that blood supply of maxillary sinus can play a role in such a result. Further studies are needed to explore whether the use of membrane could really be advantageous for the sinus augmentation procedure and to evaluate what influence this method can have on the amount and quality of reconstructed bone.

Piglet model for studying esophageal regrowth after resection and interposition of a silicone stented small intestinal submucosa tube.

PURPOSE: To investigate the use and subsequent healing of a silicone stented small intestinal submucosa (SIS) tube as a full-circumference replacement following surgical resection of the esophagus in piglets. MATERIAL AND METHODS: Three centimeters of the intrathoracic esophagus was replaced with a silicone stented SIS tube (Cook Medical) in 6 growing piglets. The esophageal stent was retained for 4 weeks. Esophageal dilations were performed, if needed, after stent removal. RESULTS: The piglets were sacrificed 1-17 weeks after surgery. Recurrent dilations were needed after stent removal. Histology showed that the gap between the resection margins was filled with new loose connective tissue consisting of fibroblasts and few inflammatory cells. In this tissue, intense angiogenesis was seen at the early time points, which then gave way to the proliferation of immature-looking smooth-muscle-like cells in the submucosa, which appeared to stem from the pericytes of the ingrowing capillaries. CONCLUSIONS: Through using a stented SIS tube as a circumferential esophageal replacement in a piglet model, this study suggests that pericytes from ingrowing capillaries may play a role in the remodeling of the SIS mesh. It remains to be seen if this process gives a favorable end result because stricture formation after stent removal remains a problem.

Differential locomotion of long- and short-term IL-2-activated murine natural killer cells in a model matrix environment.

Tumour infiltration by activated natural killer (A-NK) cells is a pre-requisite for tumour eradication by adoptive NK cell transfer. Extravasated A-NK cells do not always succeed in reaching the crucial target cell conjugation. Therefore, we wished to study A-NK cell locomotion and interactions with melanoma cells in a matrix environment (Matrigel) by electron, confocal and fluorescence microscopy. Two distinct patterns of A-NK cell-mediated matrix disintegration were revealed during incubation of tumour cells and A-NK cells in Matrigel: (1) A-NK cells pre-cultured for 5 days altered the homogeneous texture of the Matrigel, an initial microporous appearance became a loose filamentous meshwork by 24 h. Matrix degrading protease inhibitors could not fully prevent this, but could delay the process; and (2) A-NK cells pre-cultured for 6 days or more, instead formed large excavations in the Matrigel leaving the remaining matrix less affected compared to the effects by the younger A-NK cells. By histochemical staining with Cupromeronic Blue, the excavations were shown to contain proteoglycan material. Protease inhibitors had no discernable effect on the development of the excavations. The conspicuous capacity of A-NK cells to disintegrate extracellular matrix and the formation of large excavations seems only partially to depend on matrix-degrading proteases. Formation of extracellular proteoglycan material is suggested to facilitate A-NK cell locomotion within a matrix environment.

In vivo MR imaging of magnetically labeled human embryonic stem cells.

INTRODUCTION: Human embryonic stem cells (hES) have emerged as a potentially new therapeutic approach for treatment of heart and other diseases applying the concept of regenerative medicine. A method for in vivo visualization and tracking of transplanted hES would increase our understanding of in vivo hES behavior in both experimental and clinical settings. The aim of this study was to evaluate the feasibility of magnetic labeling and visualization of hES with magnetic resonance imaging (MRI). METHODS: hES were established and expanded according to standard procedures. After expansion, the cells were cultured under feeder free conditions and magnetically labeled by addition of dextran-coated Ferrum-oxide particles (Endorem) to the medium. Accumulation of small particles of iron-oxide (SPIO) in hES was assessed by Prussian blue staining and electron microscopy. For in vitro MRI, the labeled and unlabeled hES were examined in cell solution and after transplantation into explanted mouse heart ( approximately 100,000 cells) on a Bruker Avance DMX 500 vertical magnet at 11.75 T. A multi-slice, multi spin-echo T(2)-weighted images were obtained. For in vivo imaging, the experiments were performed on male Sprague-Dawley using Bruker Biospec 2.35 T magnet. The hES were directly injected ( approximately 500,000 cells) after surgical procedure (thoracotomy) into anterior left ventricular (LV) wall. Multi-slice T(2)-weighted gradient echo images were obtained using cardiac gating. RESULTS: hES appeared to be unaffected by magnetic labeling and maintained their ability to proliferate and differentiate. No additive agent for membrane permeabilisation was needed for facilitation of intracellular SPIO accumulation. Prussian blue and electron microscopy have revealed numerous iron particles in the cytoplasm of hES. On T(2)-weighted images, the labeled cells have shown well-defined hyopintense areas at the site of injection in anterior LV wall both in vitro and in vivo. CONCLUSIONS: It is feasible to magnetically label and visualize hES both in vitro and in vivo. MR visualization of magnetically labeled hES may be a valuable tool for in vivo tracking of hES.

Morphological appearance, content of extracellular matrix and vascular density of lung metastases predicts permissiveness to infiltration by adoptively transferred natural killer and T cells.

We have recently shown that adoptively transferred, IL-2-activated natural killer (A-NK) cells are able to eliminate well-established B16-F10.P1 melanoma lung metastases. However, some B16-F10.P1 lung metastases were resistant to infiltration by the A-NK cells and also resistant to the A-NK cell treatment. The infiltration-resistant (I-R) B16-F10.P1 metastases had a unique "compact" morphology compared to the "loose" morphology of the infiltration-permissive (I-P) metastases. Here, we show that I-P loose tumors and I-R compact tumors are also found in lung metastases of mouse Lewis lung carcinoma (3LL), MCA-102 sarcoma, and MC38 colon carcinoma as well as rat MADB106 mammary carcinoma origin. Furthermore, the infiltration resistance of the compact tumors is not restricted to A-NK cells, since PHA and IL-2 stimulated CD8+ T-cells (T-LAK cells) also infiltrated the compact tumors poorly. Analyses of tumors for extracellular matrix (ECM) components and PECAM-1(+) vasculature, revealed that the I-R lesions are hypovascularized and contain very little laminin, collagen and fibronectin. In contrast, the I-P loose tumors are well-vascularized and they contain high amounts of ECM components. Interestingly, the distribution pattern of ECM components in the I-P loose tumors is almost identical to that of the normal lung tissue, indicating that these tumors develop around the alveolar walls which provide the loose tumors with both a supporting tissue and a rich blood supply. In conclusion, tumor infiltration by activated NK and T cells correlates with the presence of ECM components and PECAM-1(+) vasculature in the malignant tissue. Thus, analysis of the distribution of ECM and vasculature in tumor biopsies may help select patients most likely to benefit from cellular adoptive immunotherapy.

Clonal populations of chondrocytes with progenitor properties identified within human articular cartilage.

The aim of the present study was to identify and characterize progenitor properties of human articular chondrocytes selected by using agarose suspension culture. In this chondrogenic selective culture condition, about 3.6% of seeded surplus chondrocytes from patients undergoing articular chondrocyte transplantation proliferated and formed cell clusters after 6 weeks. Phase-contrast microscopy and transmission electron microscopy revealed four different types of cell clusters differing in cellular content and matrix production. Based on their morphological features, they were named the homogenous (H), the homogenous matrix (HM), the differentiated matrix (DM) and the differentiated (D) cell clusters. All cell clusters showed positive safranin O staining, and matrix was positive for antibodies detecting type II collagen and aggrecan. The clusters were further demonstrated to express the genes for fibroblast growth factor receptor 3, type IIA collagen and type IIB collagen, while type X collagen was not expressed. After subcloning, the H and HM clusters demonstrated the best proliferative capacity. Chondrocytes from these two cell clusters also showed phenotypic plasticity in chondrogenic, adipogenic as well as osteogenic assays. This study demonstrates that existing subpopulations of cells with chondroprogenitor properties can be isolated from human adult articular cartilage using agarose suspension cultures.

Brånemark Novum: prosthodontic and dental laboratory procedures for fabrication of a fixed prosthesis on the day of surgery.

PURPOSE: The purpose of this report is to describe a new technique to fabricate and deliver an implant-supported fixed prosthesis to the patient on the day of surgery, and to propose a protocol for the prosthodontic and dental laboratory procedures. MATERIALS AND METHODS: The development of the Brånemark Novum prosthodontic protocol is reviewed, and clinical and dental laboratory assessments and methods are described. RESULTS: The total treatment time to fabricate a permanent implant-supported fixed prosthesis in the mandible can be reduced to 1 day with the Brånemark Novum technique. The new method includes a precise surgical technique using drilling templates for predetermined implant positions, a rigid splinting of the implants immediately after placement, the use of a prefabricated titanium framework, and elimination of implant impression procedures. CONCLUSION: With the technique described in this report, it is possible to provide patients with a permanent implant-supported fixed prosthesis in the mandible on the day of implant surgery. Patient benefits are obvious, with drastically reduced total treatment time, lower cost, and fewer clinical visits.

5-Fluorouracil induces autophagic degeneration in rat oral keratinocytes.

In this study, we investigated the effect of 5-fluorouracil (5-FU) on the keratinocytes of oral epithelium. Female Lewis rats were given 5-FU i.v. and were killed 12, 24 or 36 h after injection. The buccal mucosa was dissected. The number of nuclei with DNA strand breaks and the total number of nuclei per volume of the epithelial basal cell layer was estimated using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling. Epithelial cells were analysed by flow cytometry, transmission electron microscopy and a dye exclusion test. The number of cells with DNA strand breaks increased in 5-FU treated rats. Flow cytometry showed a decrease in cell size and an increase in granularity with increasing doses of 5-FU. Dye exclusion gave no indication of degenerate cell membranes. By transmission electron microscopy, the cells showed evidence of degeneration, shrinkage and loss of cell-to-cell contact. Vacuolation was extensive and, in contrast to apoptotic cells, nuclear chromatin condensation seemed to occur centrally in the nuclei. The results show that 5-FU treatment in vivo induces alterations in rat oral keratinocytes that are consistent with autophagic degeneration.

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.

Tumor structure and extracellular matrix as a possible barrier for therapeutic approaches using immune cells or adenoviruses in colorectal cancer.

In this article we report about the role that tumor structure and extracellular matrix (ECM) may play in immunotherapy and in gene therapy using adenoviruses. We performed studies in a rat model for colorectal cancer, CC531, and in specimens of human colorectal cancer. The tumors were composed of two compartments, tumor cell nests surrounded by stromal cells. ECM proteins were expressed in the stromal part, where the blood vessels were also located. Furthermore, in several tumors, the tumor cell nests were surrounded by basal membrane-like structures. Therefore, in vascular approaches to treat cancer, therapeutic agents on their route to tumor cells may be hampered by ECM to reach tumor cells. We found that immune cells were abundantly present in tumors from colorectal origin. These cells were, however, not found in direct contact with tumor cells, but mainly in the stromal part of the tumor. Adenoviruses, when intravascularly injected, did not reach tumor cells in the CC531 rat model. Tumor cells were only infected, and even then in limited numbers, in cases of intratumoral injection. We hypothesize that ECM in a tumor is a barrier both for immune cells and for adenoviruses to make direct contact with these tumor cells, and thus limits colorectal tumor therapy.

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.

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.

2B4 stimulation of YT cells induces natural killer cell cytolytic function and invasiveness.

2B4 is a surface molecule found on all human natural killer (NK) cells, a subset of CD8+ T cells, monocytes and basophils. It was originally identified on mouse NK cells and the subset of T cells that mediate non-major histocompatibility complex (MHC)-restricted killing. Recently,9 we have cloned the human homologue of 2B4 (h2B4) and found h2B4 to also mediate non-MHC-restricted cytotoxicity. In this study, we examine h2B4 in regulating various functions of NK cells using a human NK cell line YT, with monoclonal antibody (mAb) C1.7, an antibody that specifically recognizes h2B4. Ligation of surface 2B4 with mAb C1.7 increases YT's ability to destroy tumour cells. In the presence of mAb C1.7, the production of interferon-gamma (IFN-gamma) by YT cells is greatly enhanced. Engagement of surface 2B4 by mAb C1.7 downregulates the expression of h2B4 at the cell surface as well as the expression of h2B4 mRNA. Also, signalling through h2B4 causes the increased expression of matrix metalloproteinase-2, a member of the matrix degrading proteinase family. Thus, in addition to modulating cytolytic function and cytokine production of NK cells, activation through surface 2B4 may play a role in upregulating the machinery for degradation of extracellular matrices to promote invasion of the tumour by NK cells.

Secreted and membrane-associated matrix metalloproteinases of IL-2-activated NK cells and their inhibitors.

We have previously documented that rat IL-2-activated NK (A-NK) cells produce matrix metalloproteinase-2 (MMP-2) and MMP-9. In this study, we describe mouse A-NK cell-derived MMPs, including MT-MMPs, and also TIMPs. RT-PCR analysis from cDNA of mouse A-NK cells revealed mRNA for MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP, TIMP-1, and TIMP-2. MMP-2 and MMP-9 expression was confirmed by gelatin zymography. Moreover, we report for the first time that MT-MMPs are expressed by NK cells, i.e., large granular lymphocytes as determined by both RT-PCR and Western blots. TIMP-1 expression was detected as a 29-kDa protein in Western blots. It is intriguing that TIMP-2 protein from A-NK cells was also detected as a 29-kDa protein, which is clearly different from the previously reported molecular mass of 21 kDa in mouse and human cells. In addition, inhibition of MMPs by BB-94, a selective inhibitor of MMP, significantly inhibited the ability of mouse A-NK cells to migrate through Matrigel, a model basement membrane. Taken together, these findings suggest that A-NK cells may therefore use multiple MMPs in various cellular functions, including degradation of various extracellular matrix molecules as they extravasate from blood vessels and accumulate within cancer metastases following their adoptive transfer.

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.

Biodistribution and tumor localization of lymphokine-activated killer T cells following different routes of administration into tumor-bearing animals.

PURPOSE: The efficiency of adoptive cellular immunotherapy of cancer might depend on the number of effector cells that reach the malignant tissues. In the present study, the biodistribution and tumor localization of ex vivo lymphokine-activated T killer (T-LAK) cells was investigated. METHODS: T-LAK cells were labeled with 125I-dU or the fluorescent dye tetramethylrhodamine isothiocyanate (TRITC) and transferred by intravenous, -cardiac, -portal or -peritoneal injection into normal (C57BL/6) mice or mice with syngeneic day-7 to day-12 B16 melanoma metastases established in various organs. The overall biodistribution of the T-LAK cells was measured by gamma counting and their tumor localization by fluorescence microscopy. RESULTS: At 16 h after intravenous injection, the organ distribution of 125I-dU-labeled T-LAK cells was identical in normal and tumor-bearing animals. Fluorescence microscopy of lung tissue from animals receiving TRITC-labeled T-LAK cells revealed, however, a fivefold higher accumulation of T-LAK cells in lung metastases than in the surrounding normal lung tissue (1174 and 226 cells/mm2 respectively). Some pulmonary metastases were, however, resistant to infiltration. Very few intravenously injected cells redistributed to other organs or to tumors in these, since only 60 and 30 T-LAK cells/mm2 were found within metastases of the adrenal glands and the liver respectively. However, following injection of T-LAK cells via the left ventricle of the heart, a threefold increase (from 60 to 169 cells/mm2) in the number of transferred cells in metastases of the adrenal glands was observed. Moreover, following locoregional administration of T-LAK cells into the portal vein, tenfold higher numbers (from 30 to 400 cells/mm2) were found in hepatic metastases than were observed following intravenous or intracardiac injection. In the liver, a surprisingly large number of intraportally injected T-LAK cells (approx. 1300/mm2) were observed to accumulate in the perivascular spaces of the portal, but not the central veins. Even though some superficial ovarian and liver metastases were separated from the peritoneal cavity by only the peritoneal lining, no localization into these metastases was seen following intraperitoneal injection of the T-LAK cells. While treatment of tumor-bearing animals with T-LAK cells plus IL-2 reduced lung metastases by 76% as compared to treatment with IL-2 alone (P<0.03), no significant reduction of liver metastases was seen. CONCLUSIONS: T-LAK cells are able to localize substantially into tumor metastases in various anatomical locations, but mainly following locoregional injection. This finding might have important implications for the design of future clinical protocols of adoptive immunotherapy based on T cells.

The microscopic anatomy of experimental rat CC531 colon tumour metastases: consequences for immunotherapy?

The colon adenocarcinoma cell line CC531 was adopted as a model for immunotherapeutical treatment of experimental colorectal metastases in a syngeneic rat model. We studied the presence and localization of T and natural killer cells, vessels and matrix proteins in in vivo growing CC531 tumours by immunohistochemistry. CC531 tumours were induced either in the lungs by injecting CC531 tumour cells into a tail vein or in the liver by injection of CC531 tumour cells under the liver capsule or into a mesenteric vein. All 3 tumour types were composed of islets of tightly apposed tumour cells surrounded by abundantly present tumour-stroma which contained tumour vessels and matrix proteins. Some of these matrix proteins, especially laminin and collagen IV formed a basal membrane-like structure around the tumour nodules. This structure was most pronounced in mesenteric vein-induced liver tumours and less prominent in subcapsular-induced liver tumours and tail vein-induced lung tumours. Tumour-infiltrating lymphocytes of both T and natural killer cell origin were found in the tumours, but predominantly in the tumour stroma, separated from the islets of tumour cells by the basal membrane-like structure. We hypothesize that the matrix proteins of these tumours play an ambivalent role: they may provide a substratum for migration of effector cells into the tumour stroma but may also provide a barrier preventing direct contact between tumour target cells and immune effector cells.

Endogenous and adoptively transferred A-NK and T-LAK cells continuously accumulate within murine metastases up to 48 h after inoculation.

In murine models, therapeutic efficacy of adoptive immunotherapy (AIT) of cancer with lymphokine activated killer (LAK) cells is seen only when applied together with substantial doses of interleukin-2 (IL-2), probably because this cytokine is imperative for both motility and viability of the LAK cells. We wanted to investigate whether IL-2 in addition mediates an immunostimulatory activation and expansion of endogenous effector cells contributing to tumor regression. Using an immunoperoxidase technique, we have been able to longitudinally analyze the accumulation of tumor infiltrating lymphocytes expressing the pan-T cell/activated lymphocyte phenotype (Thy1.2), the natural killer (NK) cell phenotype (AsGM,) as well as the cytotoxic T (CD8) cell phenotype within experimental established B16 pulmonary melanoma metastases in C57BL/6 mice during the first 48 h after high dose IL-2 monotherapy. Whereas a substantial and selective infiltration of AsGM1+ lymphocytes in tumor tissue was seen (262 and 937 cells per sq.mm malignant tissue at 0 and 48 h, respectively), only a minor increase in accumulation of CD8+ cells was seen (106 and 171 cells per sq.mm tumor tissue at 0 and 48 h, respectively). The addition of adoptive transfer with lymphokine-activated adherent NK (A-NK) cells to the high-dose IL-2 treatment resulted in more than a 1.5 fold increase in infiltrating AsGM1+ cells compared to IL-2 therapy alone (1520 compared to 937 AsGM1+ cells per sq.mm malignant tissue). No substantial accumulation of CD8+ cells was observed in this setting either. In contrast, the treatment with high dose IL-2 together with adoptive transfer of mitogen-stimulated, lymphokine-activated T killer (T-LAK) cells increased the infiltration of CD8+ cells 10-fold compared to IL-2 monotherapy (2078 compared to 171 CD8+ cells per sq.mm malignant tissue, respectively). Interestingly, infiltration of both endogenous and exogenous cells continued over time, since the effector-to-tumor cell ratio in metastatic tissue dramatically increased from 1:8 and 1:6 at 16 h to 1:3 and 1:2 at 48 h after adoptive transfer of A-NK and T-LAK cells, respectively. These data underline the longevity of LAK cells in vivo and highlight the importance of IL-2 treatment in recruiting endogenous immune cells to tumor areas.

Characteristics of tumor infiltration by adoptively transferred and endogenous natural-killer cells in a syngeneic rat model: implications for the mechanism behind anti-tumor responses.

Interleukin-2-activated, cultured NK cells (A-NK) cells were adoptively transferred into a syngeneic rat liver-tumor model. The kinetics of tumor infiltration by NK cells, originating either from adoptively transferred or from endogenous sources, the localization of these cells in the tumor, and their interactions with extracellular-matrix proteins were studied by immunohistochemistry and transmission-electron microscopy. The adoptive transfer of A-NK cells via the hepatic artery and s.c. injections of IL-2 into rats bearing subcapsularly induced CC531 liver tumors, but also IL-2 monotherapy, resulted in a significant increase of the number of NK cells both at the tumor border and in the tumor center. The majority of tumor-infiltrating NK cells was present in the tumor stroma and only occasionally was an NK cell observed in a tumor nodule in direct contact with tumor cells. Observations by electron microscopy suggested that matrix proteins, abundantly present in the tumor stroma but absent in the tumor nodules, provide a substrate for migration of infiltrating cells, whereas tight structures of matrix proteins surrounding tumor nodules provide a barrier for establishment of direct NK-cell-to-tumor-cell-contact. Our results suggest that direct NK-cell-to-target-cell-contact-mediated lysis is of minor importance for attaining an anti-tumor effect in this model. We hypothesize that treatment of tumor-bearing rats with A-NK cells and/or IL-2 initiates a cascade of events (e.g., secretion of tumor-killing cytokines and/or infiltration of other immune cells) ultimately leading to tumor regression.

Infiltration patterns of short- and long-term cultured A-NK and T-LAK cells following adoptive immunotherapy.

Direct contact between lymphokine-activated killer (LAK) cells and tumour cells is believed to be imperative for initiating tumour cell lysis in vitro as well as in vivo. In order to optimize adoptive immunotherapy (AIT) it is therefore desirable to identify the LAK cell subtype, which ensures maximal infiltration of tumours as well as a substantial cytotoxic reactivity. In this report we have compared short- and long-term cultured murine adherent natural killer (A-NK) cells and mitogen-stimulated, lymphokine-activated T-killer (T-LAK) cells with respect to their proliferative potential, cytotoxicity, requirement for interleukin-2 (IL-2) and ability to infiltrate B16 pulmonary metastases following adoptive transfer. We found that short-term (5 days) cultured A-NK and T-LAK cells both showed a substantial accumulation of tumour tissues. However, A-NK cells gradually lost this ability during in vitro culture whereas T-LAK cells cultured for as long as 20 days retained their ability to infiltrate metastases as efficiently as their short-term cultured counterparts. Moreover, the low requirement of IL-2 by T-LAK cells to achieve maximal infiltration of tumours sharply contrasted with the excessive doses necessary to ensure maximal infiltration by A-NK cells. In conclusion, these data demonstrate that short-term cultured LAK cells of both NK- and T-cell origin are able to infiltrate B16 pulmonary metastases effectively. Importantly, the T cells retain this ability for a considerably longer time and require much less IL-2 support than do A-NK cells, making T-LAK cells attractive for AIT.