CIK as therapeutic agents against tumors.

Cytokine Induced Killer (CIK) cells are ex vivo expanded and activated T lymphocytes obtained by sequential incubation of Peripheral Blood Mononuclear cells (PBMNC) with Interferon γ (IFNG), anti CD3 monoclonal antibody OKT3 and IL2. These cells, while retaining few characteristics of the Effector memory T cells subpopulation, acquired during culture CD56 expression, as well as non specific, Natural Killer like, anti tumoral cytotoxicity. CIK cells from human are equivalent to expanded NKT cells in mouse. More interestingly, CIK cells show a potent intratumoral homing in several experimental models, followed by anti tumoral clinical activity in mice and humans. In spite of extensive in vivo permanence and proliferation, CIK cells do not show cytotoxicity against normal targets and, particularly important, do not show Graft versus host disease when tested in allogeneic combinations (donor versus host) even in the haploidentical matching. For the easiness of the laboratory preparations, the availability of clinical grade reagents, the production of Good Manufacturing Practice compliant methods, CIK cells have been extensively used for the treatment of cancer patients, in both hematologic and solid tumors, in both autologous and allogeneic combinations. Several clinical protocol will be here discussed and summarised to show the feasibility of these passive transfer approaches, and also their very limited toxicity. Finally, preliminary indications on clinical efficacy, particularly in hematologic malignancies and against minimal residual disease, will be shown and discussed, as well as the future perspectives to optimize this adoptive passive cell immunotherapy strategy by gene transfer technology or bispecific monoclonal antibodies addition.

First Report of a Successful Pregnancy in an Everolimus-Treated Heart-Transplanted Patient: Neonatal Disappearance of Immunosuppressive Drugs.

The use of everolimus (EVL) as primary immunosuppression is steadily increasing in heart transplantation (HTx) patients. Limited data currently exist in kidney transplantation, but there is no report of EVL use during pregnancy after HTx and its pharmacokinetics in the newborn. We report a case of an unplanned pregnancy discovered at 21 weeks of gestation in a female HTx patient aged 40 years treated with EVL and cyclosporine (CyA). Because pregnancy was advanced, immunosuppression therapy was left unchanged. At 36 weeks, a healthy infant was delivered. At birth, CyA blood levels were lower in the neonate, but EVL concentrations in maternal and neonatal umbilical blood were similar. Amniotic fluid concentrations were undetectable for both drugs. In the newborn, EVL was measurable at 5 days after birth, whereas CyA disappeared within 2 days. Cord blood displayed a normal count of B and T cells and CD4, CD8 and natural killer cell populations. At birth, both mother and newborn displayed the same blood levels of EVL; therefore, a filter effect of the placenta may be hypothesized for CyA but not for EVL. No immediate complications were observed with this pregnancy.

Influence of substituting dietary soybean for air-classified sunflower (Helianthus annuus L.) meal on egg production and steroid hormones in early-phase laying hens.

Soybean meal (SBM) is the most widely and expensive protein source used in the formulation of poultry diets; however, when the price of SBM increases, poultry nutritionists seek alternative sources that are more economical in formulating least-cost rations. This research aimed to evaluate the effects of dietary air-classified sunflower meal (SFM) on some productive parameters and plasma steroid hormones in laying hens. In this trial, 20-week-old laying hens (ISA Brown strain) in the early phase of production were randomly assigned to two groups and fed wheat middlings-based diets containing soybean (135 g/kg; 48% CP) or air-classified SFM (160 g/kg; 41% CP) as the main protein source. Laying performance, egg size and feed conversion ratio were evaluated for 10 week. Plasma steroid hormones (progesterone and oestradiol) in the hens were quantified weekly. Substituting SBM with air-classified SFM did not change (p > 0.05) the hens' growth performance, whereas feed consumption and efficiency were positively influenced (p < 0.05) by SFM treatment. Egg production rate was improved in hens fed the SFM diet (p < 0.05), as well as the percentage of medium-size eggs that was higher for SFM treatment (p < 0.05). Steroid hormones levels were affected by dietary treatment (p < 0.01). From our findings, it could be effective to include air-classified SFM in early-phase laying hen diets as an alternative protein source substituting SBM, without negative influence on productive performance and egg traits, reducing also the production costs.

Low-fiber alfalfa (Medicago sativa L.) meal in the laying hen diet: effects on productive traits and egg quality.

This study was designed to determine the effects on laying performance and egg quality resulting from partial substitution of soybean meal (SBM) with low-fiber alfalfa (LFA; Medicago sativa L.) meal in the diet of early-phase laying hens. ISA Brown layers, 18 wk of age, were randomly allocated to 2 dietary treatments and fed for 10 wk. The hens were fed 2 wheat middling-based diets: a control diet, which contained SBM (15% of diet), and a test diet containing LFA (15% of diet) as the main protein source. Low-fiber alfalfa meal was obtained by a combination of sieving and air-classification processes. Feed intake was recorded daily, and egg production was calculated on a hen-day basis; eggs from each group were weekly collected to evaluate egg components and quality. The partial substitution of SBM with LFA had no adverse effect on growth performance of early-phase laying hens. Egg production and none of the egg-quality traits examined were influenced by dietary treatment, except for yolk color (P < 0.001) and yolk percentage (P < 0.05) as well as yolk cholesterol and ß-carotene contents (P < 0.001), which were improved in hens fed the LFA diet. Including LFA increased serum ß-carotene and reduced serum cholesterol concentrations (P < 0.001). Our results suggest that partially replacing conventional SBM as protein source with low-fiber alfalfa meal in the laying-hen diet can positively influence yolk quality without adversely affecting productive traits.

Localization of mesenchymal stromal cells dictates their immune or proinflammatory effects in kidney transplantation.

Multipotent mesenchymal stromal cells (MSC) have recently emerged as promising candidates for cell-based immunotherapy in solid-organ transplantation. However, optimal conditions and settings for fully harnessing MSC tolerogenic properties need to be defined. We recently reported that autologous MSC given posttransplant in kidney transplant patients was associated with transient renal insufficiency associated with intragraft recruitment of neutrophils and complement C3 deposition. Here, we moved back to a murine kidney transplant model with the aim to define the best timing of MSC infusion capable of promoting immune tolerance without negative effects on early graft function. We also investigated the mechanisms of the immunomodulatory and/or proinflammatory activities of MSC according to whether cells were given before or after transplant. Posttransplant MSC infusion in mice caused premature graft dysfunction and failed to prolong graft survival. In this setting, infused MSC localized mainly into the graft and associated with neutrophils and complement C3 deposition. By contrast, pretransplant MSC infusion induced a significant prolongation of kidney graft survival by a Treg-dependent mechanism. MSC-infused pretransplant localized into lymphoid organs where they promoted early expansion of Tregs. Thus, pretransplant MSC infusion may be a useful approach to fully exploit their immunomodulatory properties in kidney transplantation.

The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity.

The membrane attack complex of complement (C) in sublytic concentrations stimulates endothelial cells (EC) to express adhesion molecules and to release biologically active products. We have examined the ability of a cytolytically inactive form of this complex, which is incapable of inserting into the cell membrane, to upregulate the expression of adhesion molecules and of tissue factor (TF) procoagulant activity. The inactive terminal C complex (iTCC) was prepared by mixing C5b6, C7, C8, and C9 and was purified by fast protein liquid chromatography on a Superose 12 column. Binding of this complex to EC was found to be dose dependent and was inhibited by anti-C9 antibodies, as assessed both by ELISA using an mAb anti-C9 neoantigen and by measuring cell-bound 125I-labeled iTCC. Exposure of EC to iTCC resulted in a dose- and time-dependent expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 accompanied by increased levels of the corresponding mRNA, but not in the rapid expression of P-selectin. Inactive TCC also induced increased TF activity evaluated by a chromogenic assay that measures the formation of factor Xa. These effects were inhibited by anti-C9 antibodies. The data support the conclusion that iTCC may induce proinflammatory and procoagulant activities on EC.

Cloning and characterization of a new isoform of the interleukin 1 receptor antagonist.

By reverse transcriptase polymerase chain reaction on messenger RNA from human polymorphonuclear cells, we have isolated a sequence identical to the cDNA coding for intracellular interleukin 1 receptor antagonist (icIL-1ra), but containing an additional in-frame 63-bp sequence located three codons downstream of the translation start of icIL-1ra. This additional sequence is inserted between the first and second exon of the intracellular form, the latter of which is colinear with part of the first exon of the secreted form of IL-1ra. The additional sequence is coded by an extra exon located 2 kb downstream the first icIL-1ra-specific exon. The complementary DNA sequence of the alternatively spliced form of icIL-1ra shows that the predicted protein differs from classical icIL-1ra in the NH2 terminus by insertion of a leaderless sequence of 21 amino acids rich in glycine and glutamic acid residues. Transcripts coding for this new form of icIL-1ra were detected in activated fibroblasts, keratinocytes, and at low levels in myelomonocytic cells. The recombinant protein expressed in COS cells had an apparent molecular mass in sodium dodecyl sulfate polyacrylamide gel electrophoresis of 25 kD compared to 22 kD of classical icIL-1ra, and was mostly intracellular. The ability of this new form of icIL-1ra to inhibit IL-1 activity, in terms of induction of E-selectin and human immunodeficiency virus replication, was comparable to that of classical icIL-1ra. We propose to refer to this new form of icIL-1ra as icIL-1ra type II.

Inhibition of interleukin-1 responsiveness by type II receptor gene transfer: a surface "receptor" with anti-interleukin-1 function.

The hypothesis that the type II receptor (RII) acts as a decoy for interleukin-1 (IL-1) was tested by gene transfer in cells expressing only the type I receptor (8387 fibroblasts). RII-transfected cells showed defective responsiveness to IL-1 in terms of NFkappaB activation, cytokine gene expression and production. Blocking monoclonal antibodies against RII restored the capacity of RII-transfected cells to respond to IL-1 beta. Hence defective IL-1 responsiveness of RII-transfected cells requires surface expression of the molecule. RII-transfected cells showed normal responsiveness to TNF, which shares functional properties and elements in the signal transduction pathway with IL-1. Cells transfected with a deletion mutant of RII missing 26 of 29 amino acids of the cytoplasmic portion of the molecule showed impaired responsiveness to IL-2. Cells transfected with full-length or the cytoplasmic deletion mutant of RII released copious amounts of RII in the supernatant. However, transfected cells showed defective responsiveness to brief exposure to IL-1, in the absence of measurable released RII. These results indicate that impairment of the responsiveness to IL-1 following RII gene transfer was dependent upon surface expression of the molecule, specific for IL-1 and unaffected by truncation of the cytoplasmic portion. Thus, the type II "receptor" is a decoy surface molecule, regulated by antiinflammatory signals, whose only known function is to capture and block IL-1.

The histone deacetylase inhibitor ITF2357 has anti-leukemic activity in vitro and in vivo and inhibits IL-6 and VEGF production by stromal cells.

We have investigated the activity of ITF2357, a novel hydroxamate histone deacetylase inhibitor, on multiple myeloma (MM) and acute myelogenous leukemia (AML) cells in vitro and in vivo. ITF2357 induced apoptosis in 8/9 MM and 6/7 AML cell lines, as well as 4/4 MM and 18/20 AML freshly isolated cases, with a mean IC(50) of 0.2 microM. ITF2357 activated the intrinsic apoptotic pathway, upregulated p21 and downmodulated Bcl-2 and Mcl-1. The drug induced hyperacetylation of histone H3, H4 and tubulin. When studied in more physiological conditions, ITF2357 was still strongly cytotoxic for the interleukin-6 (IL-6)-dependent MM cell line CMA-03, or for AML samples maximally stimulated by co-culture on mesenchymal stromal cells (MSCs), but not for the MSCs themselves. Interestingly, ITF2357 inhibited the production of IL-6, vascular endothelial growth factor (VEGF) and interferon-gamma by MSCs by 80-95%. Finally, the drug significantly prolonged survival of severe combined immunodeficient mice inoculated with the AML-PS in vivo passaged cell line already at the 10 mg/kg oral dose. These data demonstrate that ITF2357 has potent anti-neoplastic activity in vitro and in vivo through direct induction of leukemic cell apoptosis. Furthermore, the drug inhibits production of growth and angiogenic factors by bone marrow stromal cells, in particular IL-6 and VEGF.

Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts.

We compared two protocols for the expansion of human mesenchymal stromal cells (hMSCs) starting from diagnostic samples of BM aspirates (2-5 ml) or using the remnants in the bag and filter at the end of the BM infusions. The protocols differed in the presence of either 10% fetal bovine serum (FBS) or 5% platelet lysate (PL). We obtained a significantly (P=0.02) better expansion with PL, obtaining a median 1010-fold compared to 198-fold with a selected batch of FBS and in fewer days (29.8 in PL versus 41.4 in FBS). Overall, we recovered a variable number from 54.8 x 10(6) to 365 x 10(6) hMSCs in PL versus a variable number from 2.7 x 10(6) to 31 x 10(6) in FBS. No difference could be found in terms of gross morphology, differentiation potential, surface markers and immunological properties (inhibition of allogeneic PHA response and mixed lymphocyte reaction) of cells expanded with PL or FBS. The preparations were found within the range of acceptability for all the quality control criteria. Due to the clinical grade nature of the PL and the reproducibility of separate preparations, we propose this method to obtain hMSCs even from minute amounts of BM cells.

Rapid and massive expansion of cord blood-derived cytokine-induced killer cells: an innovative proposal for the treatment of leukemia relapse after cord blood transplantation.

We have used a standardized 21-day expansion protocol to produce cytokine-induced killer (CIK) cells starting from very small amounts of nucleated cells (approximately 15 x 10(6) cells) isolated from cord blood. Mononuclear cells are stimulated with anti CD3 (OKT3) and IFNgamma and then expanded with IL-2. Moreover, we show that washouts of cord blood units bags (at the end of the infusion) may be sufficient to yield almost 500 x 10(6) CIK by the same expansion protocol. CIK cells show strong cytotoxic activity against a variety of tumor target cell lines including B and T lymphomas and myeloid leukemias. More importantly, expanded cord blood-derived CIK cells are cytotoxic against fresh leukemic blasts and express perforin, granzyme and NKG2D molecule at high levels. The same in vitro protocol has already been used to expand CIK cells from peripheral blood of adult donors under GMP conditions and therefore these observations open up the possibility of imagining a future clinical application of leukemia relapse following cord blood transplantation with CIK cells obtained from the same cord blood unit.

Inhibition of natural killer activity by tumor-associated lymphoid cells from ascites ovarian carcinomas.

Tumor-associated lymphocytes (TAL) were isolated from ascites ovarian tumors by stepwise application of density and velocity sedimentation on discontinuous Ficoll-Hypaque gradients and fetal bovine serum. TAL had low levels of natural killer (NK) activity compared to levels of peripheral blood lymphocytes (PBL) from the same patients or control subjects. When TAL were mixed with PBL, significant inhibition of NK activity was observed in 7 of 27 patients tested, with the suppression levels ranging from 14 to 60%. Inhibition of PBL NK activity was observed at the ratio of TAL to PBL of 1:1 or 2:1. Suppression of NK activity was detected with effector-to-target cell ratios ranging from 6:1 to 25:1 and at incubation time from 4 to 20 hours in the cytolysis assay. Tumor-associated macrophages from 6 patients were tested for suppression of NK activity. Only with 1 donor was a 17% inhibition observed at a ratio of macrophages of PBL of 1:1. Thus suppression by mature plastic adherent macrophages does not play a major role in the determination of the low levels of NK in human ascites ovarian tumors and the inhibitory activity of suppressor TAL, which had a minor contamination (less than 5%) with mononuclear phagocytes. When TAL from 1 patient with suppressive activity were passed through nylon wool, inhibition of NK activity was observed with both adherent and nonadherent cells.

Defective natural killer activity within human ovarian tumors: low numbers of morphologically defined effectors present in situ.

Tumor-associated lymphocytes (TAL) were isolated from 17 ascites and 7 solid ovarian carcinomas. TAL had defective natural killer (NK) activity against K562. Large granular lymphocytes, the morphologically identified effectors of NK activity, were poorly represented in TAL from ovarian carcinomas as compared to peripheral blood lymphocytes from the same patients or from normal donors. Similar results were obtained when effectors of NK activity were identified with an anti-NK (HNK-1) monoclonal antibody. When four TAL preparations were separated on discontinuous Percoll gradients, they were able to be enriched for NK activity and large granular lymphocyte morphology in the lower density fractions as observed with blood. These observations suggested that a low concentration of the relevant effector cells was the major factor determining the defective NK cytotoxicity of lymphoid cells associated with these human neoplasms.

Interferon effect on cytotoxicity of peripheral blood and tumor-associated lymphocytes against human ovarian carcinoma cells.

A study was done to investigate the tumoricidal activity of peripheral blood lymphocytes (PBL) and tumor-associated lymphoid cells (TAL) against freshly isolated tumor cells in human ovarian carcinoma. TAL and carcinoma cells were purified by density and velocity sedimentation on discontinuous Ficoll-Hypaque gradients and fetal bovine serum. Purified carcinoma cells from 23 ascitic and 3 solid tumors were used as targets in a 4- or a 20-hour 51Cr release assay. K562 cells were used to measure natural killer (NK) activity. Freshly purified ovarian carcinoma cells were relatively resistant to lysis by normal unstimulated PBL. Cytolytic activity of ovarian cancer PBL and TAL did not exceed that of control PBL: Only one PBL preparation had high levels of cytotoxicity (36.2 and 42.9% specific lysis after 4 and 20 hr at an effector-to-target cell ratio of 50:1) against autologous cancer cells but not against allogeneic targets (less than 5% specific lysis). TAL and, to a lesser extent, ovarian cancer PBL showed impaired NK activity against K562 compared to the activity seen in controls. In vitro exposure to partially purified human fibroblast interferon (IFN) (1,000 U/ml for 1-18 hr) augmented NK activity against K562 of PBL and TAL. When ovarian carcinoma cells were used as targets, IFN enhanced the cytotoxicity of normal PBL in a 4- and 20-hour assay; stimulation by IFN was less frequently observed with ovarian cancer PBL and TAL in a 4-hour assay, but after 20 hours effector cells from tumor-bearing subjects showed IFN-boosted cytotoxicity against carcinoma cells similar to the degree of cytotoxicity seen in controls. IFN enhanced the cytotoxicity of PBL and TAL against both autologous and allogeneic carcinoma cells. Thus PBL and TAL are rarely cytotoxic against 51Cr-labeled fresh autologous carcinoma cells, but in vitro exposure to IFN induced low levels of killing of ovarian tumor cells.

The human A-myb protein is a strong activator of transcription.

The A-myb gene belongs to the family of the c-myb proto-oncogene. We report here the cloning from a B lymphocyte cDNA library of the previously missing 3' half of the human A-myb cDNA, thus closing the previously still incomplete open reading frame. Analysis of the homologies between the different myb proteins reveals four domains of high conservation. We show, using a polyclonal rabbit antibody, that the 90 kd human A-myb protein is nuclear and that it activates transcription from the KHK-CAT reporter 6-10 times more strongly than c-myb in NIH3T3 cells. The transactivating function of A-myb depends on the presence of the myb binding site in the reporter, and on both the DNA binding and acidic domains of the A-myb protein. The bacterially expressed protein protects the myb binding sites of the reporter in footprint experiments. Binding of the A-myb protein is shown in gel retardation assays to be specific for the classical c-myb recognition sequence PyAACG/TG. In addition, like c-myb, A-myb binds more strongly to the MIM-A synthetic oligonucleotide that carries the TAACGG sequence than to the MBS-I oligonucleotide containing TAAGTG. Finally, DNA binding activity is demonstrated to require the N-terminal portion of the protein containing the three tandem repeats of amino acids conserved in all myb proteins. We have thus shown that the A-myb protein is a strong activator of transcription and that this activity depends on both the DNA-binding and acidic domains.

Isolation and characterization of the human A-myb promoter: regulation by NF-Y and Sp1.

The A-myb transcription factor shows a restricted tissue distribution and is cell cycle regulated. Furthermore its deregulation has profound effects on the growth and/or differentiation of the cells in which it is normally expressed. We have therefore characterized its promoter. A 12 kb genomic clone was isolated that comprises the first exon, part of the first intron as well as upstream regulatory sequences. Multiple transcription start sites have been identified which operate in both B lymphocytes and epithelial cells and the upsteam region was shown to have promoter, activity. The boundaries of the minimal promoter region (-183-14), of a positive upstream (-538-183) and a negative downstream regulatory region (NRE) (+83+374) have been defined. The NRE is promoter- and orientation-independent but position specific. The A-myb minimal promoter is GC-rich, does not contain any TATA box but has a functional CCAAT box. The CCAAT box and minimal promoter is highly conserved in the corresponding murine sequence. The CCAAT box efficiently binds the NF-Y complex and its mutation decreases basal promoter activity by 50%. Two Sp1 binding sites are present upstream from the CCAAT box which can bind Spl and contribute to A-myb promoter activity by 70 and 30%, respectively. The two Sp1 sites and CCAAT box together contribute to over 80% of A-myb basal promoter activity and are therefore the major regulatory elements. Finally, we show that the promoter is cell cycle regulated and that the SP1 and CCAAT elements are required for S phase induction.

PTX3, A prototypical long pentraxin, is an early indicator of acute myocardial infarction in humans.

BACKGROUND: Inflammation is an important component of ischemic heart disease. PTX3 is a long pentraxin whose expression is induced by cytokines in endothelial cells, mononuclear phagocytes, and myocardium. The possibility that PTX3 is altered in patients with acute myocardial infarction (AMI) has not yet been tested. METHODS AND RESULTS: Blood samples were collected from 37 patients admitted to the coronary care unit (CCU) with symptoms of AMI. PTX3 plasma concentrations, as measured by ELISA, higher than the mean+2 SD of age-matched controls (2.01 ng/mL) were found in 27 patients within the first 24 hours of CCU admission. PTX3 peaked at 7.5 hours after CCU admission, and mean peak concentration was 6.94+/-11.26 ng/mL. Plasma concentrations of PTX3 returned to normal in all but 3 patients at hospital discharge and were unrelated to AMI site or extent, Killip class at entry, hours from symptom onset, and thrombolysis. C-reactive protein peaked in plasma at 24 hours after CCU admission, much later than PTX3 (P<0.001). Patients >64 years old and women had significantly higher PTX3 concentrations at 24 hours (P<0.05). PTX3 was detected by immunohistochemistry in normal but not in necrotic myocytes. CONCLUSIONS: PTX3 is present in the intact myocardium, increases in the blood of patients with AMI, and disappears from damaged myocytes. We suggest that PTX3 is an early indicator of myocyte irreversible injury in ischemic cardiomyopathy.