To determine whether early acute cellular rejection (ACR) is associated with sub-optimal immunosuppression in children with liver transplants (LTx).

METHODS

Twenty-five children with primary LTx after pre-transplant rabbit anti-thymocyte globulin (rATG), and steroid-free tacrolimus (TAC) were evaluated. Mitogen-stimulated T- and B-cell responses and mixed lymphocyte response to donor and third-party antigens were performed at several time points between two consecutive TAC doses. TAC concentrations (C) associated with half-maximal effect (EC(50)) on lymphocytes was determined by pharmacodynamic equations.

RESULTS

Mean age was 7.2 +/- 6.2 years, mean time to lymphocyte function studies was 25 +/- 19 days. Acute rejection occurred at a mean interval of 31 +/- 19 days after LTx. Rejectors (n = 16) demonstrated significantly higher EC(50) of TAC for the intra-cellular IFN-gamma in T cells (p = 0.005) and its CD8+ sub-population (p = 0.027) as well as the co-stimulatory/activation receptor CD54 on B cells (p = 0.0001). The response of recipient lymphocytes to donor antigen was significantly higher in rejectors, compared with non-rejectors (p = 0.015). The patient groups demonstrated no differences in third-party MLR, or in C of TAC.

CONCLUSIONS

Independent of the amount of immunosuppressant, ACR of liver allografts in children is associated with enhanced donor-specific alloreactivity. This is accompanied by a cytotoxic T-cell sub-population with increased requirement for TAC.

The effect of ligation of CD40 on the proliferation and Ig secretion of a battery of human Ig-secreting hybridomas was examined to determine the regulatory activity of this surface molecule on B cells after initial activation. B cell hybridomas were generated by fusing activated peripheral blood B cells with SPAZ-4, a non-Ig-secreting fusion partner, and were cloned before analysis. All hybridomas expressed CD40 comparably. These hybridomas were stimulated with either recombinant baculovirus-expressed membrane-bound CD40L or a soluble murine CD40L/CD8 construct in the presence or the absence of various cytokines. Concentrations of CD40L that saturated 40 to 100% of CD40 induced initial homotypic aggregation followed by Fas (CD95)-independent apoptosis, with resultant decreases in growth and Ig secretion. Concentrations of CD40L that saturated 15 to 25% of CD40 also stimulated aggregation of all hybridomas. However, proliferation and Ig secretion of 9 of 13 IgM-secreting hybridomas, but none of 14 IgG- or IgA-secreting hybridomas, were enhanced by these concentrations of CD40L. These responses were independent of interactions mediated by the adhesion pair CD1la/CD18-CD54. These results indicate that the impact of CD40 ligation on human Ig-secreting hybridomas varies with the extent of CD40 engagement and depending on whether the hybridoma derived from an activated B cell that had previously undergone switch recombination.

A novel tumor cell line, denominated F6, was established from mutated human embryonic bone marrow mesenchymal stem cells (MSCs) which were induced by the GM-CSF and IL-4 in vitro. The characteristics of the F6 cell line, such as surface antigens, cell cycle, growth curve, gene expression, morphology, cytogenetics and tumor model were analyzed. The F6 cells were round and grew suspended in a plastic dish. The cell line has a strong self-renewal capability, was positive for CD13, CD29, CD44, but negative for CD1alpha, CD3, CD10, CD14, CD23, CD33, CD34, CD38, CD41, CD45, CD54 and HLA-DR. The surface antigens were lower than those of human embryonic MSCs. The karyotype of F6 cells was abnormal. The cell cycle included: G0/G1 phase, 52.24%; G2/M phase, 8.00%; S phase, 41.76%. After the cells had been passaged serially for more than 17 months (62 passages), their characteristics were still retained. The F6 cells resulted in tumors in SCID nude mice in vivo (8/8) and caused metastasis (3/8). The pathologic examination revealed that the tumor cells extensively invaded surrounding normal tissues such as dermis, muscular tissue, nerve tissue, adipose tissue and lymphoid tissue. F6 cell line, tumor tissues derived from F6 cells and the MSCs expressed different levels of the nucleostemin gene. These findings suggested that F6 may be a novel tumor cell line. It may provide evidence for the theory that cancer originates from stem cells, and may be useful for the investigation on safety of human MSCs in the clinical application.

Multiple myeloma is characterized by the presence of malignant plasma cells predominantly localized in bone marrow. Our prior studies have suggested that human myeloma derived-cell lines adhere specifically to fibronectin and to bone marrow stromal cells (BMSCs) via beta 1 and beta 2 integrins as well as RGD peptide, and that tumour cell to BMSC contact triggers interleukin-6 (IL-6) secretion from BMSCs. Since IL-6 is a growth factor for myeloma, adhesion may be important in paracrine IL-6 mediated tumour cell growth. We therefore examined phenotypic expression of adhesion molecules on the U266 and IM-9 human myeloma-derived cell lines using the panel of monoclonal antibodies (MoAbs) directed at adhesion molecules submitted to the Vth International Conference on Human Leukocyte Differentiation Antigens. U266 and IM-9 myeloma cell lines express mainly CD29, CD49d, VLA-1, CD18, CD54, ICAM-2 and ICAM-3. In contrast, CD49b, VLA-3, CD49f, CD11b, VCAM-1, selectins and selectin-ligands were not expressed on these cell lines. Specific adherence of IM-9 cells to BMSC line LP101 was demonstrated which could be partially blocked by pre-incubation and culture of tumour cells with anti-beta 1 integrin, anti-beta 2 integrin, anti-CD49d, anti-VLA-5, anti-CD11a, anti-CD44 and anti-CD54 MoAbs. The combination of these MoAbs (anti-CD29, CD18, CD11a, CD49d, VLA-5, CD44, CD54, ICAM-2, ICAM-3 MoAbs) decreased but did not completely abrogate binding of IM-9 to BMSCs. Moreover, increases in IL-6 secretion from BMSCs after adherence of IM-9 cells were also partially blocked by these MoAbs. These findings suggest that multiple adhesion pathways may mediate adherence of myeloma cell lines to BMSCs, localizing tumour cells in the marrow microenvironment and triggering IL-6 secretion by BMSCs which may augment tumour cell growth.

Three types of trivalent influenza vaccines were analysed for their in vitro stimulatory properties on immune cells from young healthy volunteers. A whole inactivated virus (WV) vaccine, a conventional subunit (c-SU) preparation and a new virosomal subunit (v-SU) vaccine were used. Blood-derived DC up-regulated MHC class II, CD54, CD80 and CD86 after exposure to WV vaccine, indicating their functional maturation, but were only moderately affected by subunit (SU) vaccines. In addition, IL-12 and tumour necrosis factor-alpha (TNF-alpha) secretion by DC were markedly enhanced by WV, but not by SU vaccines. The production of IL-2 and interferon-gamma (IFN-gamma) by PBMC was also strongly stimulated by WV, but much less by SU vaccines, among which the v-SU vaccine was a better stimulator of IL-2 secretion. In contrast to WV vaccine both SU vaccines were powerful stimulators of PBMC proliferation. Our results suggest that the presence of influenza core components leads to the activation of DC and triggers the production of cytokines by PBMC. SU vaccines are in contrast excellent stimulators of T cell growth. A combination of WV and SU vaccines in immunization regimes might allow optimal T cell priming as well as the efficient generation and maintenance of memory cells.

Contact hypersensitivity (CHS) has been widely used to study cutaneous immune responses, as a prototype of delayed-type hypersensitivity. Although natural killer T (NKT) cells have been assumed to have an important role in CHS, their role is controversial. Here, we report the role of NKT cells in the sensitization phase of CHS, by promoting the survival and maturation of dendritic cells (DCs) in the draining lymph nodes (LNs). The CHS response was attenuated with Cd1d1(-/-) and Traj18(-/-) BALB/c mice in which NKT cells were absent. In the draining LNs, the number of effector T cells and cytokine production were significantly reduced with NKT cell-deficient mice. NKT cells activated and colocalized with DCs in the draining LNs after sensitization. The number of migrated and mature DCs was reduced in NKT cell-deficient mice 72 hours after FITC application. In in vitro experiments, activated NKT cells enhanced bone marrow-derived DC (BMDC) survivability via tumor necrosis factor (TNF) production from BMDCs. In addition, TNF production from BMDCs was partially suppressed by the neutralizing anti-CD54 or CD154 antibodies. Our data demonstrate that DC-NKT interaction has a pivotal role in the sensitization phase of CHS.

We examined a possible role for the adhesion molecules LFA-1 and ICAM-1 in localizing central nervous system non-Hodgkin's lymphomas (CNS-NHLs) to the brain. Fresh frozen sections from 12 monoclonal CNS NHLs (11 primary, one secondary) were stained with monoclonal antibodies to LFA-1 alpha chain (CD11a), beta chain (CD18) and, ICAM-1 (CD54). Additional staining made use of rat monoclonal antibodies to the human and mouse high endothelial venule antigens HECA 452 and MECA 79 and mouse ICAM-1. The expression of these same molecules was also studied in mice with severe combined immunodeficiency (SCID) mice, bearing intracranial human lymphoblastoid cells. Eleven of the CNS-NHL tumors expressed LFA-1 alpha (one strongly, one intermediate, nine weakly). Nine of the tumors weakly expressed LFA-1 beta.. Nine of twelve tumors weakly expressed ICAM-1. In six of seven tumors definite blood vessels stained for ICAM-1. Non-tumor brain from two patients and non-tumor cerebral blood vessels showed no staining with CD11a, CD18 or CD54 antibodies. Strong expression of LFA-alpha and LFA-beta as well as ICAM-1 was noted in human lymphoblastoid cells (LCLs)/SCID mouse CNS lymphomas. Tumor blood vessels in these mice stained for mouse ICAM-1. Normal SCID mouse brains showed no staining with CD11a, CD18, CD54 or mouse ICAM-1 antibodies. Human, human/mouse CNS lymphomas, normal human, and mouse brains showed no staining with either HECA 452 or MECA 79.(ABSTRACT TRUNCATED AT 250 WORDS)

Intravascular large cell lymphoma (IVLL) is a rare neoplasm characterized by a proliferation of lymphoma cells within the blood vessels. Its cell origin and clinicopathological characteristics have not been well understood. The study uses 5 male and 4 female patients who were diagnosed as having IVLL from 1978 to 1996. We examined cell lineage and adhesion molecules using immunohistochemical staining and performed a molecular analysis by using polymerase chain reaction (PCR) on the IgH gene, on T-cell receptor chain genes, and the Epstein-Barr virus (EBV) and in situ hybridization on EBV. The immunohistochemical and PCR results disclosed 8 cases of B- cell and one of T-cell lymphoma. Three of four cases whose frozen specimens were available expressed CD5. PCR showed EBV in 7 of 9 cases, although EBV was found by in situ hybridization in only 3 cases. Lymphoma cells express CD11a and CD49d (VLA-4), while endothelial cells expressed CD54 (CD11a ligand) and CD106 (CD49d ligand). Such interaction of these adhesion molecules might contribute to the intravascular proliferation of lymphoma cells. Furthermore, the CD5 expression of lymphoma cells suggests that IVLL most likely originates from a unique subtype of B cells, although their normal counterpart remains uncertain.

OBJECTIVE

To investigate the expression of and monokine induction by interleukin 18 (IL-18; also called interferon-gamma inducing factor, IGIF), in peripheral blood mononuclear cells (PBMC) and cultured synoviocytes from rheumatoid arthritis (RA) patients.

METHODS

We carried out IL-18 Western blotting and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) of cytokines in PBMC [IL-18, IL-1beta and tumour necrosis factor alpha (TNF-alpha)] and long-term cultured fibroblast-like synoviocytes (FLS) [IL-18, IL-1beta, TNF-alpha, IL-6, interferon gamma (INF-gamma) and [granulocyte-macrophage colony stimulating factor (GM-CSF)] from RA patients and controls. FLS were isolated from RA synovial membranes (FLS(SM)) and RA synovial fluids (FLS(SF)), osteoarthritis (OA) FLS(SM) and FLS(SF) from spondyloarthropathy patients. FLS were characterized by fluorescence-activated cell sorting of the FLS. PBMC and FLS from RA patients and control subjects were stimulated with recombinant human IL-18 and IL-1beta (rHuIL-18/rHuIL-1beta), and TNF-alpha, IL-1beta and MMP-1 were measured by ELISA in supernatants.

RESULTS

Constitutive expression of IL-18 mRNA was significantly reduced whereas that of TNF-alpha was enhanced in RA PBMC. Persistent low expression of IL-18, TNF-alpha, GM-CSF and IL-1beta was observed in RA and OA FLS(SM) as well as spondyloarthropathy FLS(SF). In contrast, high constitutive expression of IL-18 in FLS (CD90/Thy-1- and CD54-positive, CD14- and CD86-negative), accompanied by persistent high levels of TNF-alpha, GM-CSF and IL-1beta expression, was restricted to synovial fluid-derived FLS obtained from RA patients. IFN-gamma was not detectable in any culture, but IL-6 mRNA was equally expressed in all FLS cultures. rHuIL-18 was effective in stimulating TNF-alpha and IL-1beta secretion in PBMC from healthy controls, but failed to stimulate TNF-alpha and IL-1beta secretion from PBMC in 11 of 12 RA patients, and all FLS cultures. rHu-IL-1beta, but not rHu-IL-18, induced interstitial collagenase (MMP-1) in FLS.

CONCLUSIONS

Persistent high production of proinflammatory cytokines in RA-FLS(SF) may be relevant for chronic progression in RA synovitis. Levels of TNF-alpha and IL-1beta expression are increased in RA-FLS(SF), but are independent of IL-18. The pathological function of enhanced IL-18 expression in RA-FLS(SF) remains to be further elucidated.

BACKGROUND

Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication of allogeneic bone marrow transplantation (BMT). IPS is associated with elevated bronchoalveolar lavage (BAL) fluid levels of tumor necrosis factor-alpha and lipopolysaccharide, both of which are potent activators of endothelial cells (ECs). EC expression of the adhesion molecule CD54 (intercellular adhesion molecule [ICAM]-1) has been shown to be a major regulator of pulmonary inflammation in various experimental models.

METHODS

Using a well-established murine BMT system in which lung injury and graft-versus-host disease (GvHD) are induced by minor histocompatibility antigenic differences between donor and host, the RNase Protection Assay, mice deficient in ICAM-1 expression, and a monoclonal blocking antibody to ICAM, we evaluated the role of the pulmonary vascular expression of CD54 in the development of IPS.

RESULTS

Enhanced pulmonary vascular expression of ICAM-1 coincided with the development of IPS. When ICAM-1 -/- mice were used as allogeneic BMT recipients, IPS severity (measured by lung histopathology, BAL cellularity, and cytokine expression) was significantly reduced compared with wild-type controls. Similar results were also observed when wild-type recipients were treated with a monoclonal blocking antibody to ICAM-1. Surprisingly, ICAM-1 had differential effects on leukocyte infiltration into GvHD target organs; ICAM-1 deficiency had no impact on intestinal histopathology, whereas ICAM-1-/- BMT recipients had significantly enhanced hepatic injury.

CONCLUSIONS

These data demonstrate that although the expression of ICAM-1 is critical for the development of IPS, different mechanisms of leukocyte recruitment are operative in other GvHD target organs.

Interleukin (IL)-4 is thought to contribute to the Th2 type of immune response and hence the development of allergic reactions such as asthma. In asthmatic patients, the airway epithelium expresses increased amounts of the cell surface adhesion molecule intercellular adhesion molecule (ICAM)-1 (CD54). One cytokine capable of inducing ICAM-1 in airway epithelial cells, tumor necrosis factor-alpha (TNF-alpha), is present in asthma. This study evaluated if IL-4 either alone or together with TNF-alpha costimulation might modulate CD54 expression by human bronchial epithelial cells (HBECs). CD54 positivity increased in response to IL-4 (16 +/- 2% positive vs. 3 +/- 1%, P < 0.01); greater induction of CD54 resulted from TNF-alpha (45 +/- 2%, P < 0.001). Costimulation with TNF-alpha plus IL-4 further augmented expression (56 +/- 1%, P < 0.05). Immunoperoxidase results were confirmed by flow cytometry. RT-PCR revealed no increase in ICAM-1 mRNA expression under control conditions or after stimulation with IL-4 alone. TNF-alpha increased IL-4 mRNA, and IL-4 potentiated this. Functionally, IL-4 augmented the adhesion of THP-1 monocyte/macrophage cells to monolayers of HBECs both alone and in the presence of TNF-alpha. We conclude that 1) IL-4 augments epithelial cell ICAM-1 expression, 2) IL-4 potentiates the adhesion of THP-1 monocyte/macrophage cells to epithelial cells, and 3) modulation of epithelial cell ICAM-1 expression by IL-4 may play a role in the immunopathology of bronchial asthma.

Human thioredoxin (Trx) is the major 12-kd cellular disulfide-reductase that on secretion acts as a cocytokine with several interleukins. Truncated Trx with the 80 N-terminal residues (Trx80), also present in plasma, was by itself a mitogenic cytokine for human peripheral blood mononuclear cells (PBMC). This study investigated which cells in PBMC are targets of recombinant Trx80. Purified human CD14(+) monocytes, but not B or T cells, in a synthetic medium were activated to differentiation by Trx80 as measured by flow cytometry of surface antigens because exposure to 100 nM Trx80 increased expression of CD14, CD40, CD54, and CD86. Proliferation of the monocytes was increased in a dose-dependent manner by Trx80 in concentrations ranging from 10 nM to 1 microM. Trx or interleukin (IL) 2 did not induce proliferation or expression of surface antigens on monocytes. Trx80 alone induced secretion of IL-12 from CD40(+) monocytes in the PBMC cultures and this effect was enhanced by IL-2. Trx80 and IL-2 together were strongly synergistic to induce secretion of interferon-gamma in PBMC cultures. The results showed that Trx80 is a potent cytokine for normal human monocytes and directs the immune system in favor of a Th1 response via IL-12 production.

Normal skin of healthy individuals and both lesional and uninvolved skin from patients with psoriasis before and after receiving cyclosporin A (CsA; 2.5 or 5 mg/kg per day) was examined by immunocytochemistry for differences in expression of adhesion-relevant epitopes. Normal, lesional and uninvolved skin all showed staining of basal keratinocytes for CD29 (the common beta chain of the beta 1-integrin family). No other adhesion molecule investigated was detected on structural components of normal skin. In uninvolved skin, weak expression of CD54 (intercellular adhesion molecule 1, ICAM-1) was noted on vascular endothelium. Uninvolved keratinocytes were found to stain with anti-CD58 (leucocyte function-associated antigen 3, LFA-3) and there was weak expression of CD11b (alpha chain of complement C3bi receptor) and CD11c (alpha chain of p150, 95 molecule) but not CD11a (leucocyte function-associated antigen 1, LFA-1, alpha chain) on those cells. In lesional skin, in addition to expression of CD58, there was also enhanced expression of CD11c. Weak expression of CD54 on keratinocytes was also observed. Lesional blood vessels were found to stain strongly with anti-CD54, CD29 and CD58. CD11a was expressed only on infiltrating mononuclear cells. CsA treatment produced marked clinical improvement, accompanied by the loss of CD54 expression on keratinocytes. However, despite the loss of T cells from lesional skin with CsA treatment, CD54 persisted on blood vessels. CsA was found to have no effect on keratinocyte expression of CD29, CD58 or CD11b and c. The persistence of CD54 on vascular endothelium and of adhesion molecule expression on keratinocytes, despite resolution of the skin lesions, may explain the universal and rapid recurrence of psoriasis on cessation of CsA administration.

Regulation of the avidity of LFA-1 (CD11a/CD18, alpha L beta 2) for its ligand ICAM-1 (CD54) was studied in human B cells by evaluating the effects of a phorbol ester, anti-IgM antibodies, staurosporine, and okadaic acid. We monitored changes in LFA-1 avidity by quantifying binding of cells to an immobilized rICAM-1 fusion protein. In this assay, the protein kinase C-activating phorbol ester PDB and anti-IgM antibodies, as well as the protein kinase inhibitor, staurosporine, were able to induce LFA-1-dependent binding to ICAM-1. This demonstrates that the high avidity state of LFA-1 can be induced by a protein kinase C-dependent and by a protein kinase C-independent pathway. Furthermore, treatment of the cells with the protein phosphatase inhibitor, okadaic acid, inhibited binding to ICAM-1. Treatment with staurosporine before addition of okadaic acid not only induced enhanced binding of cells to ICAM-1, but also dramatically reduced the ability of okadaic acid to inhibit binding. These results suggest a critical role for a protein phosphatase in inducing the high avidity state of LFA-1 as well as a role for a protein kinase in inducing the low avidity state of LFA-1.

CD4, a lymphocyte surface glycoprotein, serves as co-receptor for antigen with the T cell receptor (TCR). It is also the lymphocyte receptor for HIV by binding the gp120 viral envelope protein. Interaction of gp120 with CD4 is crucial for viral infection, but is not sufficient to allow viral entry into cells. Recombinant gp120 alters CD4+ T cell responsiveness to activation stimuli. To express its co-receptor function fully, CD4 must be laterally associated with the TCR and CD45 to form multi-receptor complexes competent to transduce potent activation signals. Here, we examine the possibility that gp120/CD4 binding alters lateral associations of CD4 with other lymphocyte surface molecules, and that assembly of abnormal multi-molecular complexes is involved in the gp120-induced CD4+ T cell dysfunction and in viral entry. In the absence of gp120, CD4 displayed high association with CD3, CD5, CD45RC, CD25, CD28, CD44, and CD53; weak association with CD2, CD38, CD45RB, CD62L, and CD26; and no association with CD45RA, CD45RO, CD11b, CD11a, CD54, CD7, CD48, CD98, CD59 CD55, HLA class I and class II molecules. Treatment with gp120 significantly increased CD4 association with CD3, CD45RA, CD45RB, CD59, CD38, CD26 and HLA class I, and decreased that with CD45RC. Specificity of these results were assessed at various levels. First, gp120 did not influence lateral associations displayed by other molecules, such as HLA class II. Second, the Leu3 mAb which binds CD4 on a site overlapping the gp120 binding site, did not elicit the same CD4 lateral associations as gp120, and finally, a direct gp120/CD4+ interaction was needed to induce the lateral associations, as shown by the observation that blocking the gp120/CD4 binding by the Leu3 mAb inhibited the gp120-induced associations. These results can be interpreted in several ways gp120/CD4 interaction could trigger an inside-out signal responsible for the associations, or gp120 could induce steric modifications of CD4 that increase its affinity for the associating molecules. Alternatively, these molecules may interact directly with gp120, bridging them with CD4. It is also possible that th e associations may be mediated by additional components, interacting with both gp120 and the associating surface molecule. The last hypothesis is likely for CD59, whose gp120-induced association with CD4 required the presence of serum in the co-capping assay. Since both CD59 and gp120 bind complement, the observed association could be mediated by complement components.

Lipid rafts are plasma membrane microdomains rich in cholesterol, sphingolipids, and cell surface receptors. Recent studies demonstrated the upregulation and localization of two receptors, intercellular cell adhesion molecule-1 (ICAM, CD54) and endothelial leukocyte adhesion molecule-1 (E-selectin, CD64E), within lipid raft microdomains of inflamed or injured endothelial cells (ECs). We hypothesized that the localization of ICAM and E-selectin within lipid rafts may be essential for drug delivery vehicles labeled with antibodies against ICAM (aICAM) and E-selectin (aE-selectin). To eliminate localization of cell surface receptors, ECs were treated with a cholesterol depleting drug, methyl-β-cyclodextrin. We also tested if antibody mobility and the ratio of aICAM to aE-selectin on immunoliposomes influenced binding to lipid-raft-depleted cells. Liposomes were prepared from either 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC, C(18:1), T(m) = -20 °C) or 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, C(16:0), T(m) = 42 °C) which are in the liquid crystalline and gel phase at 37 °C, respectively. Mobility and the aICAM:aE-selectin ratio influenced cellular binding only when lipid rafts form. In the absence of lipid rafts, cellular binding of both DOPC and DPPC immunoliposomes was reduced to the nonspecific binding level. These results, which were obtained under static conditions, suggest that the presence of lipid rafts in ECs is critical for targeted drug delivery.

Dendritic cells (DC) are promising candidates for cancer immunotherapy. However, it is not known whether in vitro-generated monocyte-derived DC from cancer patients are altered compared with DC from healthy donors. In a clinical phase I/II study, monocyte-derived DC were generated in vitro utilizing granulocyte macrophage colony-stimulating factor and rh-interleukin-4 (IL-4) and used for cancer immunotherapy. In this study, we tested the effect of various maturation cocktails and performed a comparative evaluation of the DC phenotype and functional characteristics. Polyriboinosinic polyribocytidylic acid (Poly I:C) + tumour necrosis factor-alpha (TNF-alpha) induced significant IL-12 p70 secretion, which was increased after addition of a decoy IL-10 receptor. The lymph node homing chemokine receptor CCR-7 expression was induced by TNF-alpha + IL-1beta + IL-6 + prostaglandin E2 but was not induced by Poly I:C + TNF-alpha. In general, DC from patients had an intermediate maturity phenotype with a significantly higher expression of CD40 and CD54 compared with healthy donors. In vitro analyses showed an unimpaired capacity of the patient-derived DC for antigen-specific (cytomegalovirus, tetanus and keyhole limpet haemocyanin) T-cell stimulation, whereas the allostimulatory capacity of patient-derived DC was significantly decreased. These data suggest that patient-derived DC are more differentiated but are less sensitive to maturation-inducing agents than DC obtained from healthy individuals.

This study systematically evaluated the conditions required for generating immature rat bone marrow-derived dendritic cells (BMDCs) and characterized their phenotype. The culture of Wistar rat bone marrow cells for 7 days in an optimal cytokine environment (granulocyte macrophage-colony stimulating factor (GM-CSF), 10 ng/ml; IL-4, 5 ng/ml) resulted in adherent and non-adherent cell populations, but only the adherent population predominantly expressed the rat DC marker OX62. Adherent OX62+ cells were immature, in that they expressed lower levels of CD86 and MHC class II and were more phagocytic than their non-adherent OX62+ counterparts. Adherent BMDCs constitutively produced low levels of IL-12 and nitric oxide (NO), levels of both of which were markedly increased following lipopolysaccharide (LPS) activation. Activation also increased the proportion of OX62+ cells expressing CD40, CD54 and CD86 and their intensity of expression, however, unlike murine BMDCs, it had no effect on CD80 and MHC class II expression. Although the proliferation of allogeneic Lewis splenocytes in response to immature resting and LPS-activated (mature) Wistar BMDCs was of a similar magnitude, levels of IL-12 after 5 days were significantly higher in cultures containing LPS-activated BMDCs and the IFN-gamma/IL-4 cytokine ratio differed markedly (2.35 vs. 6.66, respectively). This study systematically defines conditions for generating immature rat BMDC populations and demonstrates qualitative differences in the phenotype of immune responses induced by resting and LPS-activated BMDC populations.

Cancer stem cells (CSCs) are considered one of the key contributors to chemoresistance and tumor recurrence. Therefore, the precise identification of reliable CSC markers and clarification of the intracellular signaling involved in CSCs remains a great challenge in fields relating to cancer biology. Here, we implemented a novel chemoresistant prostate cancer patient-derived xenograft (PDX) model in NOD/SCID mice and identified CD54 as a candidate gene among the most highly enriched gene expression profiles in prostate tumors exposed to chronic cisplatin administration. Additional in vitro and in vivo assays showed that CD54 played a critical role in the self-renewal and tumorigenesis of prostate CSCs. Moreover, silencing CD54 greatly reduced the tumorigenesis of prostate cancers both in vitro and in vivo and significantly extended the survival time of tumor-bearing mice in a prostate cancer xenograft model. Dissection of the molecular mechanism revealed that the p38-Notch1 axis was the main downstream signaling pathway in CD54-mediated regulation of CSCs in prostate cancers. Together, these results established that CD54 could be a novel reliable prostate CSC marker and provided a new potential therapeutic target in prostate cancer via CD54-Notch1 signaling.

Rhabdomyosarcomas (RMS) are rare and often lethal diseases. It is assumed that the tumor microenvironment (TME) of RMS exerts an immunosuppressive function, but there is currently no systematic analysis of the immune cells infiltrating sarcoma tissue. Focusing on two common types of RMS (alveolar [RMA] and embryonal [RME]), we performed a comprehensive immunohistochemical analysis of tumor-infiltrating immune cells in the TME. We performed a qualitative estimation of infiltrating immune cells in the tumor microenvironment by an experienced pathologist as well as a quantitative digital pathology analysis. We found that (1) manual and automatic quantification of tumor-infiltrating immune cells were consistent; (2) RME tumors showed a higher degree of immune cell infiltration than RMA tumors but (3) the number of tumor infiltrating lymphocytes was low compared to other solid tumor types; (4) microvascular density correlated with immune cell infiltration and (5) CD163 positive macrophages as well as CD54 positive microvessels were more often detected in RME than in RMA and correlated with patient overall and event free survival. Our systematic analysis provides a comprehensive view of the immune landscape of RMS which needs to be taken into account for developing immunotherapies for this rare type of cancer.

We have retrospectively studied the diagnostic and predictive value of immunohistochemical characterization of adhesion molecules (ICAM-1, CD54, VCAM-1) and HLA-DR antigen in a homogeneous clinical group of 36 patients. Between 1 January 1991 and 31 January 1993, 130 patients received a kidney transplant in our unit. Biopsies of renal allografts were only performed in asymptomatic patients who had graft dysfunction, revealed by an isolated serum creatinine increase. Available frozen samples were included in this study (n = 44). The 35 cases of acute rejection diagnosed by biopsy corresponded to mild acute rejection according to the Banff classification criteria. First, we compared the expression of HLA-DR, ICAM-1 and VCAM-1 to morphological data to determine if the immunohistochemical data improved the histopathological diagnosis when the interstitial infiltrate was mild with slight tubulitis. We also studied the phenotype of infiltrating cells with monoclonal antibodies directed against T helper cells, T cytotoxic-suppressor cells, activated T cells and macrophages. Expression on tubular epithelium and density of each type of cell was graded semiquantitatively. Expression of HLA-DR, ICAM-1 and VCAM-1 was observed on tubular epithelium and endothelium in both acute rejection and other causes of graft dysfunction, limiting its diagnostic value. Activated T cells expressing CD69-AIM (activation inducer molecule) and/or HLA-DR were frequently observed in acute rejection (24/35 (69%) and 25/35 (71%) respectively) but not in other causes of renal dysfunction. We then studied the prognostic usefulness of the immunohistochemical profile in acute rejection. Of 27 patients, 12 had a progressively decreased renal function or returned to dialysis within one year after transplantation while the other 15 had a stable graft function after at least 18 months of follow-up. In the group of bad prognosis (n = 12), corticosteroid-resistant rejection episodes were significantly more frequent (p < 0.01). In this group, nine patients had an overexpression of HLA-DR on tubular epithelium versus one patient in the group of stable graft function (chi 2c = 10.57, p < 0.002). Seven patients included in the group of bad prognosis showed tubular overexpression of both ICAM-1 and VCAM-1 versus one patient in the other group chi 2c = 6.23, p < 0.02). Moreover, patients of the first group had a significantly higher number of interstitial macrophages as compared with those who had stable graft function (chi 2c = 4.87, p < 0.01). Thus, our data show that the immunohistochemical profile studied is of little value in the diagnosis of renal allograft rejection. However, an intense tubular expression of HLA-DR and/or both ICAM-1 and VCAM-1, and a high number of interstitial macrophages are significantly related to unfavorable graft outcome.

Recent studies have suggested that ICAM-1 (CD54) is involved in the pathogenesis of human multiple myeloma. A monoclonal antihuman CD54 antibody has been generated by immunizing BALB/c mice with human myeloma cell lines. SCID mice injected with human ARH-77 myeloma cells develop disseminated myeloma which is similar in several respects to multiple myeloma in humans. The mice have monoclonal gammopathy and succumb to hind leg paralysis caused by infiltration of tumor cells into the thoracolumbar vertebrae, resulting in compression of the spinal cord. In the absence of treatment, the mean paralysis time of the SCID/ARH-77 mice is 29 days. When the SCID/ARH-77 mice received four consecutive daily i.v. injections of anti-CD54 mAb commencing 1 day after tumor inoculation, they survived for 150 days, at which time the experiment was terminated. Histopathological analyses indicated that prior to death all control SCID/ARH-77 mice had myeloma cells in the vertebrae and skull. At this time, the anti-CD54-treated mice had no evidence of tumor. High levels of human immunoglobulin were detected in the sera of control, but not treated mice. F(ab')2 fragments of the anti-CD54 antibody also had similar, albeit, slightly less antitumor activity in vivo, suggesting that antibody effector function may account for some, but not all the antitumor activity of anti-CD54. In vitro studies indicate that anti-CD54 does not inhibit homotypic adhesion, the binding of myeloma cells to murine bone marrow stromal cells, or cell proliferation. By exclusion, we propose that the CD54-mediated homing of these ARH-77 cells to certain anatomical sites is crucial for their growth in vivo.

CD40L, the ligand for CD40 on dendritic cells (DCs), plays an important role in their activation and is essential for induction of antigen-specific T-cell responses. In the present study, we investigated the efficacy of antitumor immunity induced by vaccination with DCs engineered to express CD40L and pulsed with Mut1 tumor peptide. Our data show that transfection of DCs with recombinant adenovirus AdV-CD40L resulted in activation of DCs with up-regulated expression of proinflammatory cytokines (IL-1beta and IL-12), chemokines (RANTES, IP-10, and MIP-1alpha), and immunologically important cell surface molecules (CD54, CD80, and CD86). Our data also demonstrate that DCs transfected with AdV-CD40L (DC(CD40L)) are able to stimulate enhanced allogeneic T-cell proliferation and Mut1-specific CD8(+) cytotoxic T-cell responses in vitro. Vaccination of mice with Mut1 peptide-pulsed control virus-transfected DC (DC(pLpA)) could only protect mice from challenge of a low dose (0.5 x 10(5) cells per mouse, 8/8 mice), but not a high dose (3 x 10(5) cells per mouse, 0/8 mice) of 3LL tumor cells. However, vaccination of Mut1 peptide-pulsed AdV-CD40L-transfected DC(CD40L) induced an augmented antitumor immunity in vivo by complete protection of mice (8/8) from challenge of both low and high doses of 3LL tumor cells. Thus, DCs engineered to express CD40L by adenovirus-mediated CD40 ligand gene transfer may offer a new strategy in production of DC cancer vaccines.

OBJECTIVE

To observe the biological specialization of human peripheral blood dendritic cells (DC) and cord blood derived DC and its effects on effector cells killing human hepatocarcinoma cell line BEL-7402. in vitro.

METHODS

The DC biological characteristics were detected with immunohistochemical and MTT assay. Two antitumor experiment groups are divided: peripheral blood DC and cord blood DC groups. Peripheral blood DC groups used LAK cells as the effector cells and BEL-7402 as target cells, while cord blood DC groups used CTL induced by tumor antigen twice pulsed DC as effector cells and BEL-7402 as target cells, additional peripheral blood DC and cord blood DC are added to observe its stimulating activities to effector cells. The effector's cytotoxicity to tumor cells were detected with neutral red colorimetric assay at two effector/target ratios of 5:1 and 10:1.

RESULTS

Peripheral blood DC and cord blood DC highly expressed HLA-ABC, HLA-DR, HLA-DQ, CD54 and S-100 protein. The stimulating activities to lymphocyte proliferation were compared between experimental groups (DC added) and control group (no DC added), in six experiment subgroups,the DC/lymphocyte ratio was sequentially 0.25:100, 0.5:100, 1:100, 2:100, 4:100 and 8:100.A values were sequentially 0.75396+/-0.009, 0.84916+/-0.010, 0.90894+/-0.012, 0.98371+/-0.007, 1.01299+/-0.006 and 1.20384+/-0.006 in peripheral blood DC groups and 0.77650+/-0.005, 0.83008+/-0.007, 0.92725+/-0.007, 1.05990+/-0.010, 1.15583+/-0.011, 1.22983+/-0.011 in cord blood DC groups. A value was 0.59517+/-0.005 in control group. The stimulating activities were higher in experimental groups than in control group (P<0.01), which were increased when the DC concentration was enlarged (P<0.01). Two differently derived DCs had the same phenotypes and similar stimulating activities (P<0.05). In peripheral blood DC groups, the cytotoxicity of the LD groups (experimental groups) and L groups (control group) was 58.16%+/-2.03% (5:1), 46.18%+/-2.25% (10:1) and 38.13%+/-1.29% (5:1) and 65.40%+/-1.56% (10:1) respectively; in cord blood DC groups, TD groups (experimental groups) and T groups (control groups) were 69.71%+/-2.33 % (5:1), 77.64%+/-1.94% (10:1) and 56.89%+/-1.82% (5:1) and 60.99%+/-1.42% (10:1) respectively.The cytotoxicity activities were enhanced with increased effector/target ratio (P<0.01). At the same effector/target ratio, the cytotoxicity of experimental groups were bigger than that of control groups (P<0.01). The cytotoxicity activities of cord blood DC groups were higher than that of peripheral blood DC groups (P<0.01).

CONCLUSIONS

Peripheral blood DC and cord blood DC are mature DC in morphology and function, both can enhance the effector cell killing activities to hepatocarcinoma cells. DC pulsed with tumor antigen can induce higher specific CTL activity than unpulsed DC.