Cell-mediated Activity Research Articles

GITR Ligand on Leukemic Myeloid Dendritic Cells Suppresses Induction of Leukemia-Associated Antigen-Specific CTLs from Naïve CD8+ T Cells.

Glucocorticoid-induced TNFR-related protein (GITR), a member of the TNF receptor superfamily, is expressed at low levels on resting T cells and is up-regulated following activation. Triggering of GITR with its ligand (GITRL) has been described to abrogate the function of CD4+CD25+ regulatory T cells and stimulate effector T cells in mice, but little is known about roles of GITR-GITRL interaction in humans. Recent evidence suggests that the interaction between GITR on NK cell and GITRL, which is constitutively expressed on tumor cells, negatively regulates NK cell-mediated anti-tumor activity in cancer patients. Leukemic myeloid dendritic cells (mDCs) can induce cyclin-dependent kinase (CDK2)- specific cytotoxic T lymphocytes (CTLs) from naïve T cells and CDK2-specific CTLs are detectable in MRD+ patients with leukemia after allo-SCT (Blood. 110 (11): a3228. 2007). The GITR-GITRL interaction may affect this process and a modification of the interaction may improve the efficiency of inducing CDK2-specific CTLs. Therefore, the expression of GITRL on leukemic cells and leukemic mDCs was examined to determine whether an engagement of GITR controls the priming of leukemia-associated-antigen (LAA)-specific CD8+ T cells. When cryopreserved BMMCs obtained at diagnosis from 5 patients with AML were assessed using flow cytometry, GITRL expression was detectable on leukemic cells in 3 patients. Leukemic mDCs were enriched from PBMCs of HLA-A24+ patients with anti-CD1c mAb-conjugated magnetic beads and were assessed for their ability to stimulate HLA-A24+ naïve CD8+ T cells to acquire cytotoxicity specific to CDK2-peptides (CDK2 158–166, CDK2 178–186). A three days culture of immature leukemic mDCs in the presence of TNFα up-regulated the expression of GITRL along with CD83 and CD40 expression. Naïve CD8+ T cells isolated from healthy individuals and cord blood were cultured with the GITRL-expressing leukemic mDCs in the presence or absence of anti- GITR monoclonal antibodies (mAbs) for 14 days and stained for CDK2 158–166/A24, CDK2 178–186/A24 pentamers. Blocking of GITR with the mAbs augmented induction of CDK2 158–166- and CDK2 178–186- specific CD8+ T cells from 0.37% to 1.17% and from 0.45% to 1.64%, respectively (Fig). Anti-GITR mAbs did not enhance induction of CDK2-specific T cells by peptide-pulsed monocyte-derived DCs which do not express GITRL. These data suggest that the expression of GITRL on circulating leukemic mDCs may suppress induction of CTLs specific to LAAs and induce cancer immunoediting in patients with leukemia. Administration of anti-GITR mAb after allo-SCT may enhance graft versus leukemia effect by CDK2-specific CTLs without vaccination of CDK2-peptides. [Display omitted]

Natural killer cells suppress full cycle HCV infection of human hepatocytes

The role of natural killer (NK) cells in controlling hepatitis C virus (HCV) infection and replication has not been fully delineated. We examined NK cell-mediated noncytolytic effect on full cycle HCV infection of human hepatocytes. Human hepatocytes (Huh7.5.1 cells) co-cultured with NK cells or treated with supernatants (SN) from NK cells cultures had significantly lower levels of HCV RNA and protein than control cells. This NK cell-mediated anti-HCV activity could be largely abolished by antibody to interferon-gamma (IFN-gamma). The investigation of the mechanisms for NK cell-mediated anti-HCV activity showed that NK SN-treated hepatocytes expressed higher levels of IFN-alpha/beta than the control cells. NK SN also enhanced IFN regulatory factor-3 and 7 expression in the hepatocytes. In addition, NK SN enhanced the expression of signal transducer and activator of transcription 1 and 2, the nuclear factors that are essential for the activation of IFN-mediated antiviral pathways. These data provide direct evidence at cellular and molecular levels that NK cells have a key role in suppressing HCV infection of and replication in human hepatocytes.

Galectin-9 expands unique macrophages exhibiting plasmacytoid dendritic cell-like phenotypes that activate NK cells in tumor-bearing mice

Galectin-9 (Gal-9) inhibits the metastasis of tumor cells by blocking their adhesion to endothelium and the extracellular matrix. In this study, we addressed the involvement of Gal-9 in anti-tumor activity. Gal-9 significantly prolonged the survival of B16F10 melanoma-bearing mice. Gal-9 increased the numbers of NK cells, CD8 T cells and macrophages in tumor-bearing mice. Gal-9-mediated anti-tumor activity was not induced in NK cell-, macrophage- and CD8 T cell-depleted mice. NK cells from Gal-9-treated mice, compared to PBS-treated mice, exhibited significantly higher cytolytic activity. Co-culture of naïve NK cells with macrophages from Gal-9-treated mice resulted in enhanced NK activity, although Gal-9 itself did not enhance the NK activity. We also found that Ly-6C +CD11b +F4/80 + macrophages with plasmacytoid cell (pDC)-like phenotypes (PDCA-1 and B220) were responsible for the enhanced NK activity. These results provide evidence that Gal-9 promotes NK cell-mediated anti-tumor activity by expanding unique macrophages with a pDC-like phenotype.

Identification of the Endostyle as a Stem Cell Niche in a Colonial Chordate

Stem cell populations exist in "niches" that hold them and regulate their fate decisions. Identification and characterization of these niches is essential for understanding stem cell maintenance and tissue regeneration. Here we report on the identification of a novel stem cell niche in Botryllus schlosseri, a colonial urochordate with high stem cell-mediated developmental activities. Using in vivo cell labeling, engraftment, confocal microscopy, and time-lapse imaging, we have identified cells with stemness capabilities in the anterior ventral region of the Botryllus' endostyle. These cells proliferate and migrate to regenerating organs in developing buds and buds of chimeric partners but do not contribute to the germ line. When cells are transplanted from the endostyle region, they contribute to tissue development and induce long-term chimerism in allogeneic tissues. In contrast, cells from other Botryllus' regions do not show comparable stemness capabilities. Cumulatively, these results define the Botryllus' endostyle region as an adult somatic stem cell niche.

Right-sided lateralisation of ovarian cancer and right bias asymmetry for involved pelvic lymph nodes by ovarian cancer cells

The aim of the present study was to investigate if there is a possible lateralisation for ovarian cancers, to re-examine left–right asymmetry in pelvic lymph nodes distribution in patients with ovarian cancer, and to investigate if pelvic lymph node involvement by metastatic invasion of ovarian cancer cells is ipsilateral or contralateral. There was right-sided lateralisation for ovarian cancer. The numbers of external iliac and hypogastric+obturator lymph nodes were higher on the right side in patients with ovarian cancer on the right side; but they were about equal for right and left sides in patients with ovarian cancer in their left side. The numbers of external iliac and hypogastric+obturator lymph nodes involved by metastatic cancer cells were higher on the right side in patients with ovarian cancer on the both right and left sides. This case may result from the stronger cell-mediated immune activity in the left sides of humans.

Essential Oil of Niaouli Preferentially Potentiates Antigen-Specific Cellular Immunity and Cytokine Production by Macrophages

In vivo immunomodulatory effect of essential oil of niaouli (EON) was investigated using a mouse model, in which mice were immunized with keyhole limpet hemocyanin (KLH) and intraperitoneally given EON (less than 500 μl kg-1 body weight). In vivo efficacy of EON for immune potentiation was convinced by significantly higher expression of an activation marker, CD25, on freshly isolated draining lymph node (LN) T cells, but not B cells. However, immunofluoresence analysis failed to show any proportional change in T/B and CD4+/CD8+ T cell ratios. Data of KLH-specific immunoglobulin serum levels showed that EON does not affect humoral immune response. Instead, proliferative response and IFNγ production of LN T cells ex vivo stimulated with KLH were significantly higher in EON-treated group, but not IL-2 and IL-4 production. These results clearly show that EON preferentially upregulates T-cell mediated cellular immunity. We further clarified the accessory cells' contribution to the EON-mediated potentiation of cellular immunity and found considerably higher production of and TNF-α and IL-12 by splenic macrophages from EON-treated mice when stimulated with lipopolysaccharide (LPS) and IFNγ. Collectively, in vivo EON treatment potentiates T cell-mediated cellular immunity and macrophage activity, but not humoral immunity. The current study provides a rationale for clinical application of EON to control infectious diseases, in particular, those caused by intracellular pathogens.

Potential for Hepatitis C Virus Resistance to Nitazoxanide or Tizoxanide

Nitazoxanide and its primary metabolite, tizoxanide, inhibit hepatitis C virus (HCV) replication in HCV replicon systems. To study the potential for resistance, we subjected Huh7 cells harboring HCV replicons to serial passage in 250 muM G418 and increasing concentrations of nitazoxanide or tizoxanide. Passage of the replicon-containing cell lines in either compound resulted in increases in the 50% effective concentrations (EC(50)s) (7- to 13-fold), EC(90)s (14- to 36-fold), and 50% cytotoxic concentrations (2- to 4-fold) of both compounds. Serial passage in either compound did not alter the susceptibility of HCV replicons to ribavirin or 2'-C-methylcytidine. Interestingly, serial passage in nitazoxanide or tizoxanide resulted in increased sensitivity to alpha interferon 2b: EC(50)s and EC(90)s were reduced three- and eightfold, respectively. Replicons isolated from these cell lines had no greater ability to confer tizoxanide resistance, or increased susceptibility to alpha interferon, than replicons isolated from the parental cell line that had not previously been exposed to nitazoxanide or tizoxanide. These findings are indicative of a cell-mediated activity differing from that of other anti-HCV drugs but complementary with interferon and are consistent with the enhanced response rates observed clinically when nitazoxanide is combined with pegylated interferon therapy. Finally, unlike data for other compounds in advanced clinical development for HCV, these data are consistent with resistance in HCV replicon-containing cell lines conferred by changes in the host and not by mutations in the virus.

Immunological tolerance induced by liver grafting in the rat: splenic macrophages and T cells mediate distinct phases of immunosuppressive activity

In the rat combination DA into PVG, liver grafts are not rejected but induce donor-specific transplantation tolerance. We have examined the immunosuppressive properties of spleen cells from PVG recipients of DA liver grafts at various times post-grafting. The results indicate the development of two phases of cell-mediated suppressor activity, which appear to be mediated by separate spleen cell populations. Mitomycin-C-treated spleen cells taken from animals between 5 and 28 days post-grafting were able to suppress rat mixed lymphocyte reactions (MLRs). These 'early' suppressor cells were glass adherent and absent from populations purified by passage through nylon wool or G10 Sephadex columns. Suppression of MLR by purified glass adherent cells was not specific for either stimulator or responder haplotypes and was blocked by indomethacin. Nylon wool purified T cells were not suppressive at this time. Spleen cell suppressor activity declined to background levels after 35 days post-grafting. However, spleen cells from long-term surviving liver graft recipients (20 weeks or more) were again able to suppress MLR; the 'late' suppressor cells were nylon wool non-adherent and suppression was specific for the donor (DA) MHC type. We conclude that liver grafting in this combination generates early and late phases of suppression among spleen cells, that the early phase is produced by macrophages and mediated by prostaglandins and that the late phase is dependent on allospecific suppressor T cells.

Month-related variability in immunological test results; implications for immunological follow-up studies

This longitudinal study was originally designed to detect changes in the in vitro immune response of healthy subjects as a result of a psychological intervention. In this study a significant proportion, about 70%, of the immunological variability in the test results was accounted for by the differences in immunological response levels of the subjects. Apart from this between-subject-effect, a significant proportion of the variability in test results was related to the month of data sampling. The month-effect was computed in such a way that the between-subject variation was taken into account. This resulted in a more accurate estimation of the month-effect. Even after correction for the intervention, i.e. the defence of the PhD thesis, the effect of month of data sampling remains significant for mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, percentage of CD4 and CD8 cells, and for the response to the mitogens phytohaemagglutinin, pokeweed mitogen and concanavalin A as well as the results for the mixed lymphocyte culture for one pool out of three. In contrast, no significant month-effect was observed for the whole blood cell counts, for the differential white blood cell counts as determined by monoclonal antibody staining for cell surface markers CD3, CD16, TAC and OKM1, nor for the immunoglobulin IgM and IgG serum levels. Likewise the cell-mediated lympholysis activities measured against three pools of stimulator cells remained unaltered. We discuss the implications for future immunological follow-up studies of the observation that a significant proportion of the variability in immunological test results is related to differences between subjects and to the month of data sampling.

Inhibition of HIV replication by CD8+ T cells correlates with CD4 counts and clinical stage of disease.

We sought to evaluate the relationship of CD8+ T cell-mediated inhibition of autologous HIV replication in vitro to disease stage in HIV+ individuals. Depletion of CD8+ T cells from peripheral blood lymphocytes of 16 HIV+ subjects increased the percentage of virus-producing cultures from 56% to 81%. CD4+ T cells were purified from 52 HIV+ individuals and cultured alone or in the presence of autologous CD8+ T cells. In 13 (25%) subjects HIV replication was only detected in the absence of CD8+ T cells (inhibition positive); in 26 (50%) viral replication occurred both in the absence and presence of CD8+ cells (inhibition negative). In the remaining 13 (25%) subjects, CD8+ T cell-mediated inhibitory activity could not be evaluated because stimulation of their purified CD4+ T cells did not result in p24 production. In some virus culture-negative individuals, the inability to demonstrate HIV replication was due to the presence of low numbers of CD8+ T cells that co-purified with CD4+ T cells. Detection of inhibitory CD8+ T cells was associated with significantly higher CD4 counts and better clinical status compared with inhibition-negative subjects. These results demonstrate that CD8+ T cell-mediated inhibition of HIV replication correlates with disease stage, and thus may play a role in preventing disease progression. CD8+ T cells did not inhibit autologous HIV replication across a semipermeable membrane. Further, the ability of CD8+ T cells to prevent HIV replication did not correlate with lysis of autologous CD4+ T cells. Thus, CD8+ T cells inhibited autologous HIV replication in vitro through a contact-mediated non-lytic mechanism.

TWIST modulates prostate cancer cell-mediated bone cell activity and is upregulated by osteogenic induction

TWIST, a helix-loop-helix transcription factor, is highly expressed in many types of human cancer. We have previously found that TWIST confers prostate cancer cells with an enhanced metastatic potential through promoting epithelial-mesenchymal transition (EMT) and a high TWIST expression in human prostate cancer is associated with an increased metastatic potential. The predilection of prostate cancer cells to metastasize to bone may be due to two interplaying mechanisms (i) by increasing the rate of bone remodeling and (ii) by undergoing osteomimicry. We further studied the role of TWIST in promoting prostate cancer to bone metastasis. TWIST expression in PC3, a metastatic prostate cancer cell line, was silenced by small interfering RNA and we found that conditioned medium from PC3 with lower TWIST expression had a lower activity on stimulating osteoclast differentiation and higher activity on stimulating osteoblast mineralization. In addition, we found that these effects were, at least partly, associated with TWIST-induced expression of dickkopf homolog 1 (DKK-1), a factor that promotes osteolytic metastasis. We also examined TWIST and RUNX2 expressions during osteogenic induction of an organ-confined prostate cancer cell, 22Rv1. We observed increased TWIST and RUNX2 expressions upon osteogenic induction and downregulation of TWIST through short hairpin RNA reduced the induction level of RUNX2. In summary, our results suggest that, in addition to EMT, TWIST may also promote prostate cancer to bone metastasis by modulating prostate cancer cell-mediated bone remodeling via regulating the expression of a secretory factor, DKK-1, and enhancing osteomimicry of prostate cancer cells, probably, via RUNX2.

Age-Related Effects of Cocultured Rat Nucleus Pulposus Cells and Macrophages on Nitric Oxide Production and Cytokine Imbalance

A study of age-related effects on nitric oxide (NO) and cytokine production in cocultured rat nucleus pulposus (NP) cells and macrophages. To evaluate the effects of age on NO and cytokine production in an in vitro model of cocultured NP cells and macrophages. It is well known that the clinical characteristics of lumbar disc herniation differ with age. The relationship between age-related differences in clinical features and immuno-chemical factors, such as NO and inflammatory cytokines, has not been established. Male Sprague Dawley rats (n = 45), including 15 animals from 3 groups (3-, 12-, and 32-weeks old), were used. NP cells and exudated peritoneal macrophages were cocultured in serum-free media. NO levels were measured at 2-, 24-, 48-, and 72 hours using the Griess method. After 7 days of culture, the production of cytokines, including tissue inhibitor metalloproteinase-1, interferon-gamma (IFN-gamma), and interleukin-10 (IL-10) were evaluated. NO levels of coculture increased with age. In the coculture groups, tissue inhibitor metalloproteinase-1 and IFN-gamma level of 3 weeks old were statistically higher than 12 and 32 weeks old. IL-10 level of 3 weeks old was statistically lower than 12 and 32 weeks old. NO levels of cocultures increased with age that suggests inflammatory reactions increase with age. This study showed an age-related cytokine imbalance, as represented by levels of IFN-gamma and IL-10. Stress and aging are thought to affect the extracellular matrix and change the immunologic response. Younger rat NP cells had higher cell-mediated immunity activity, while the older rat had higher humoral immunity activity. These results demonstrate that age affects the immunologic response attributable to NP cells. Further studies are needed to elucidate the mechanism of this newly observed occurrence and to apply these findings clinically.

HDAC inhibitor reduces cytokine storm and facilitates induction of chimerism that reverses lupus in anti-CD3 conditioning regimen

In allogeneic hematopoietic cell transplantation (HCT), donor T cell-mediated graft versus host leukemia (GVL) and graft versus autoimmune (GVA) activity play critical roles in treatment of hematological malignancies and refractory autoimmune diseases. However, graft versus host disease (GVHD), which sometimes can be fatal, remains a major obstacle in classical HCT, where recipients are conditioned with total body irradiation or high-dose chemotherapy. We previously reported that anti-CD3 conditioning allows donor CD8(+) T cells to facilitate engraftment and mediate GVL without causing GVHD. However, the clinical application of this radiation-free and GVHD preventative conditioning regimen is hindered by the cytokine storm syndrome triggered by anti-CD3 and the high-dose donor bone marrow (BM) cells required for induction of chimerism. Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) are known to induce apoptosis of cancer cells and reduce production of proinflammatory cytokines by nonmalignant cells. Here, we report that SAHA inhibits the proliferative and cytotoxic activity of anti-CD3-activated T cells. Administration of low-dose SAHA reduces cytokine production and ameliorates the cytokine storm syndrome triggered by anti-CD3. Conditioning with anti-CD3 and SAHA allows induction of chimerism with lower doses of donor BM cells in old nonautoimmune and autoimmune lupus mice. In addition, conditioning with anti-CD3 and SAHA allows donor CD8(+) T cell-mediated GVA activity to reverse lupus glomerulonephritis without causing GVHD. These results indicate that conditioning with anti-CD3 and HDAC inhibitors represent a radiation-free and GVHD-preventative regimen with clinical application potential.

Intracellular Retention of the NKG2D Ligand MHC Class I Chain-Related Gene A in Human Melanomas Confers Immune Privilege and Prevents NK Cell-Mediated Cytotoxicity

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.

Light-Dark Variations in Neopterin Serum Levels in Patients With ST-Segment Elevation Acute Coronary Syndrome Undergoing Primary Angioplasty

Activation of the cell-mediated immune system plays an important role in atherogenesis and in triggering the development of acute coronary syndrome. Neopterin, a biomarker of cell-mediated immune system activity, is also a well-known biomarker of coronary artery disease. It is known that the occurrence of coronary heart disease events is not distributed evenly throughout the day but instead undergoes cyclical variation. In addition, both the number and activity level of immune system cells also vary throughout the 24-h circadian period. We studied how the serum concentration of neopterin varies during the light and dark phases of the day in patients with ST-segment elevation acute coronary syndrome who had undergone primary angioplasty and in a group of control subjects without evidence of atherosclerotic disease. The serum concentration of neopterin was determined at 10:00 in the morning (i.e., in light) and at 03:00 at night (i.e., in darkness) in 96 patients and 84 control subjects under strictly controlled conditions of light and darkness. Significant light-dark variations were observed in the serum concentration of neopterin in both patients with ST-segment elevation acute coronary syndrome (i.e., 10.2+/-3.8 nmol/L in light vs. 8.84+/-2.22 nmol/L in darkness; P< .001) and control subjects (i.e., 6.98+/-1.96 nmol/L in light vs. 5.87+/-2 nmol/L in darkness; P< .001). Moreover, levels were significantly higher in patients (P< .001). Diurnal variations in cell-mediated immune system activity were observed in both patients with ST-segment elevation acute coronary syndrome who underwent primary angioplasty and in a control group. Moreover, activity was significantly higher in patients.

GA101, a Novel Humanized Type II CD20 Antibody with Glycoengineered Fc and Enhanced Cell Death Induction, Mediates Superior Efficacy in a Variety of NHL Xenograft Models in Comparison to Rituximab.

GA101 is a novel humanized and glycoengineered CD20 antibody that was derived by humanization of the parental B-Ly1 mouse antibody and subsequent glycoengineering leading to the following characteristics: high affinity binding to the CD20 type II epitope; low complement dependent cytotoxicity (CDC) activity; high direct cell death induction; and high antibody-dependent cellular cytotoxicity (ADCC) potency. In vivo studies examined the consequences of GA101 on the growth of aggressive NHL xenograft models in SCID beige mice and the subsequent effects on survival. These studies demonstrated an outstanding dose-dependent anti-tumor activity of GA101. In particular, GA101 mediated superior anti-tumor efficacy in staged s.c. Z138 mantle cell lymphoma (MCL) and SU-DHL4 diffuse large B cell lymphoma (DLBCL) xenograft models in direct comparison to rituximab. Weekly dosing of GA101 at 10 mg/kg (Z138 model) or 30 mg/kg (SU-DHL4) was able to induce complete tumor remission and long-term survival (cures) in all animals. Consistent anti-tumor efficacy against established tumors was also observed in a number of additional NHL models including Raji Burkitt's lymphoma or OCI-LY18 DLBCL models. In addition GA101 was evaluated in an orthotopic disseminated Z138 model. After i.v. injection of Z138 cells SCID beige mice developed large intraperitoneal lymphoid tumors in the ovary. Several experiments showed that GA101 mediated increased overall and median survival in this model in direct comparison to rituximab. All xenograft studies were performed in SCID beige mice that are incompetent for NK-mediated ADCC. Indeed, control experiments with non-glycoengineered GA101 in s.c. xenograft models suggest that the superiority of GA101 does not depend on enhanced interactions of the engineered Fc region with murine effector cells such as macrophages/monocytes or granulocytes. Thus, the anti-tumor effects of GA101 in s.c. xenograft models can be primarily attributed to non-effector cell mediated activities. Currently, experiments are ongoing to evaluate the efficacy of GA101 in combination with classical chemotherapy regimens and novel targeted agents. First results from these studies will be presented during the 2007 ASH Annual Meeting. In summary it can be assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.

Asymmetries in pelvic lymph nodes and their metastatic involvement by gynecologic cancer cells

The aims of this study were to re-examine left-right asymmetry in pelvic lymph node distribution in patients with gynecologic malignancies, and to investigate if there is a left-right asymmetry in pelvic lymph node metastatic involvement by gynecologic cancer cells. The oncologic database of our gynecologic department was reviewed to identify patients who had pelvic lymphadenectomy as part of treatment for a variety of gynecologic malignancies. Right and left lymph node counts with and without involvement of cancer cells were retrieved from the pathological reports. Three hundred and thirteen patients were included in the study. The numbers of external iliac, and hypogastric + obturator lymph nodes were higher on the right side than on the left in all gynecologic malignancies. The numbers of involved external iliac, and hypogastric + obturator lymph nodes by metastatic cancer cells were significantly higher on the right side than on the left in all gynecologic malignancies. The results suggest the existence of a left-right asymmetry in pelvic lymph node distribution and pelvic lymph node distribution involved by gynecologic cancer cells. This situation may be due to the asymmetry in the number of pelvic lymph nodes. In addition, stronger cell-mediated immune activity in the left side of humans may be associated with the blocking of metastatic invasion of cancer cells from gynecologic malignancies in the left side of the body.

Oral Administration of High Molecular Mass Poly-γ-Glutamate Induces NK Cell-Mediated Antitumor Immunity

We analyzed the in vivo tumor regression activity of high molecular mass poly-gamma-glutamate (gamma-PGA) from Bacillus subtilis sups. chungkookjang. C57BL/6 mice were orally administered 10-, 100-, or 2000-kDa gamma-PGA or beta-glucan (positive control), and antitumor immunity was examined. Our results revealed higher levels of NK cell-mediated cytotoxicity and IFN-gamma secretion in mice treated with higher molecular mass gamma-PGA (2000 kDa) vs those treated with lower molecular mass gamma-PGA (10 or 100 kDa) or beta-glucan. We then examined the effect of oral administration of 10- or 2000-kDa gamma-PGA on protection against B16 tumor challenge in C57BL/6 mice. Mice receiving high molecular mass gamma-PGA (2000 kDa) showed significantly smaller tumor sizes following challenge with the MHC class I-down-regulated tumor cell lines, B16 and TC-1 P3 (A15), but not with TC-1 cells, which have normal MHC class I expression. Lastly, we found that gamma-PGA-induced antitumor effect was decreased by in vivo depletion of NK cells using mAb PK136 or anti-asialo GM1 Ab, and that was completely blocked in NK cell-deficient B6 beige mice or IFN-gamma knockout mice. Taken together, we demonstrated that oral administration of high molecular mass gamma-PGA (2000 kDa) generated significant NK cell-mediated antitumor activity in mice bearing MHC class I-deficient tumors.

Alloantigen recognition is critical for CD8 T cell-mediated graft anti-tumor activity against murine BCL1 lymphoma after myeloablative bone marrow transplantation

The goal of the current study was to determine whether whole bone marrow cells or splenic CD8(+) T cells from C57BL/6 (H-2(b)) donor mice, which are tolerant to BALB/c (H-2(d)) alloantigens, are capable of mediating graft anti-tumor activity against a BALB/c B-cell lymphoma after injection into irradiated BALB/c hosts. The experimental results show that high doses of splenic CD8(+) T cells mixed with T cell-depleted bone marrow cells from C57BL/6 non-tolerant (normal) donors eliminate the BCL(1) B-cell lymphoma cells and induce lethal graft-versus-host disease (GVHD). CD8(+) T cells from tolerant donors simultaneously lose both their ability to induce GVHD and their anti-tumor activity. Whole bone marrow cell transplants from normal donors eliminated BCL(1) tumor cells without inducing GVHD, and bone marrow cells from tolerant donors failed to eliminate the tumor cells. The infused BCL(1) tumor cells expressed an immunogenic tumor-specific idiotype antigen disparate from host alloantigens, indicating that recognition of the tumor-specific antigen alone was insufficient to elicit graft anti-tumor activity from unimmunized allotolerant donor splenic CD8(+) T cells or whole bone marrow cells. We conclude that CD8(+) T cells from unimmunized normal donor mice require alloantigen recognition to mediate their anti-tumor activity following allogeneic BMT.

Poly(ethylene glycol) hydrogels conjugated with a collagenase-sensitive fluorogenic substrate to visualize collagenase activity during three-dimensional cell migration

We have developed collagenase-sensitive hydrogels by incorporating a collagenase-sensitive fluorogenic substrate (CS-FS) within the backbone of a polyethylene glycol (PEG) copolymer to visualize collagenase activity during three-dimensional cell migration. CS-FS was synthesized by conjugating Bodipy dyes to a peptide with collagenase-sensitive sequence, Leu-Gly-Pro-Ala (LGPA), and the products were grafted into the collagenase-sensitive PEG hydrogels. CS-FS both in solution and hydrogels had an increase in the fluorescence intensity after proteolytic degradation by collagenase, but not by non-targeted proteases nor in the absence of an enzyme. Fibroblasts inside the hydrogels conjugated with CS-FS spread and extended lamellipodia in three dimensions over several days, and their pericellular collagenase-mediated proteolysis of the hydrogel was visualized via confocal microscopy. A matrix metalloproteinase inhibitor, served as a negative control, significantly reduced the degradation rate of CS-FS by collagenase and prevented cell migration and cell-mediated collagenase activity inside these hydrogels. In summary, we have fabricated collagenase-sensitive hydrogels incorporated with CS-FS and successfully visualized the collagenase activity during three-dimensional cell migration.