Assessment of executive functioning in children and young adults treated for frontal lobe tumours using ecologically valid tests.

There is a lack of studies assessing executive functions (EF) using ecologically valid tests in children with frontal lobe lesions. This study aimed to (1) evaluate EF in children, adolescents and young adults treated for childhood frontal lobe tumours, (2) identify factors influencing performance, such as age at diagnosis or type of treatment, and (3) examine correlations between intellectual ability and classical and ecological tests of EF. Twenty-one patients, aged 8-27 years, treated for a childhood benign or malignant frontal lobe tumour, and 42 healthy controls (matched for gender, age and socio-economic status) were assessed using classical tests of EF, and the BADS-C ecological battery. Patients also underwent assessment of intellectual ability and parent and teacher ratings of the BRIEF questionnaire. IQ scores ranged from 45 to 125 (mean FSIQ = 84) and were lower in case of epilepsy, hydrocephalus and lower parental education. Patients displayed deficits in most, but not all measures of EF. Most classical and ecological measures of EF were strongly correlated to IQ. This study confirms the frequency of EF deficits in this population; it also highlights the utility of ecological measures of EF and some limitations of classical tests of EF in children.

Identification of CD4+ and CD8+ T cell epitopes of woodchuck hepatitis virus core and surface antigens in BALB/c mice.

A therapeutic vaccine against chronic hepatitis B virus (HBV) infection requires the development of a strong and multispecific Th1 cell immune response. Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) closely resemble HBV infection and represent the best animal model for this hepadnavirus-induced disease. Using the BIMAS "HLA Peptide Binding Predictions" program, we have identified and further characterized novel H-2 d-restricted CD8+ epitopes within the WHV core (peptides C#12-21, C#18-32, C#19-27, C#61-69) and surface antigens (peptides preS2#10-18, preS2#27-35, S#76-84, S#133-140 and S#257-265), respectively. These peptides bind to H-2 d with high efficiency and upon immunization of mice with peptide and Freund's adjuvant they induce the development of IFN-gamma producing T cells. More importantly, WHV core peptides C#19-27 and C#61-69 and WHV surface peptides S#133-140 and S#257-265 were also recognized by CD8+ T cells after immunization of mice with DNA/PEI nanoparticles. Direct stimulation of splenocytes obtained from such DNA-immunized mice with peptides C#18-32, S#76-84, and S#257-265 resulted in significant production of IFN-gamma. Thus, we have identified T cell determinants in mice from WHV core and surface antigens that have important value for designing and evaluating an effective vaccine against hepadnavirus infection.

Efficient recovery of HLA class I expression in human tumor cells after beta2-microglobulin gene transfer using adenoviral vector: implications for cancer immunotherapy.

Here we report a successful use of a non-replicating adenovirus expressing the wild-type human beta2m gene in recovery of normal human leucocyte antigen (HLA) class I expression in beta2m-null cancer cells. Total loss of HLA class I expression in these cell lines is caused by a mutation in beta2m gene and a loss of heterozygosity in chromosome 15 carrying another copy of that gene. Normal HLA class I expression on the tumour cell surface is critical for the successful outcome of cancer immunotherapy as T cells can only recognize tumour-derived peptides in a complex with self-HLA class I molecules. In this report we characterize the newly generated adenoviral vector AdCMVbeta2m and demonstrate an efficient beta2m gene transfer in tumour cell lines of different histological origin, including melanoma, prostate and colorectal carcinoma. The beta2m re-expression lasted for an extended period of time both in vitro and in vivo in human tumour xenograft transplants. We propose that in a subset of cancer patients with structural defect in beta2m gene or chromosome 15, the adenoviral-mediated recovery (or even increase) of HLA class I expression on tumour cells in combination with vaccination or adoptive T-cell therapy can provide a complementary approach to improve the clinical efficacy of cancer immunotherapy.

Targeting of Hsp32 in solid tumors and leukemias: a novel approach to optimize anticancer therapy.

Heat shock protein 32 (Hsp32), also known as heme oxygenase-1 (HO-1), is a stress-related anti-apoptotic molecule, that has been implicated in enhanced survival of neoplastic cells and in drug-resistance. We here show that Hsp32 is expressed in most solid tumors and hematopoietic neoplasms and may be employed as a new therapeutic target as evidenced by experiments using specific siRNA and a Hsp32-targeting pharmacologic inhibitor. This Hsp-32 targeting drug, SMA-ZnPP, was found to inhibit the proliferation of neoplastic cells with IC(50) values ranging between 1 and 50 microM. In addition, SMA-ZnPP induced apoptosis in all neoplastic cells examined. Furthermore, SMA-ZnPP was found to synergize with other targeted and conventional drugs in producing growth-inhibition. Resulting synergistic effects were observed in all tumor and leukemia cells examined. Interestingly, several of the drug partners, when applied as single agents, induced the expression of Hsp32 in neoplastic cells, suggesting that synergistic effects resulted from SMA-ZnPP-induced ablation of a Hsp32-mediated survival-pathway that is otherwise used by tumor cells to escape drug-induced apoptosis. Together, Hsp32 is an important survival factor and target in solid tumors and hematopoietic neoplasms, and may be used to optimize anticancer therapy by combining conventional or targeted drugs with Hsp32-inhibitors. Based on these data, it seems desirable to explore the value of Hsp32-targeting drugs as anti-cancer agents in clinical trials.

Targeting of mTOR is associated with decreased growth and decreased VEGF expression in acute myeloid leukaemia cells.

BACKGROUND: The mammalian target of rapamycin (mTOR) has recently been implicated in leukaemic cell growth, tumour-associated angiogenesis and expression of vascular endothelial growth factor (VEGF). We examined whether mTOR plays a role as regulator of growth and VEGF-expression in acute myeloid leukaemia (AML). Three mTOR-targeting drugs, rapamycin, everolimus (RAD001) and CCI-779, were applied. The effects of these drugs on growth, survival, apoptosis and VEGF expression in primary AML cells and various AML cell lines were examined. MATERIALS AND METHODS: Growth of AML cells and AML-derived cell lines was assessed by (3)H-thymidine incorporation, survival was examined by light- and electron microscopy, by Tunel assay and by AnnexinV-staining, and the expression of VEGF by Northern blotting, RT-PCR and ELISA. RESULTS: Rapamycin was found to counteract growth in the AML cell lines U937 and KG1a as well as in primary AML cells in 14/18 patients examined. The effects of rapamycin and its derivatives were dose-dependent (IC(50): 10 pM-100 nM). It was also found that exposure to mTOR-targeting drugs resulted in apoptosis and in decreased expression of VEGF in leukaemic cells. CONCLUSIONS: mTOR-targeting drugs exert antileukaemic effects on AML cells in vitro through multiple actions, including direct inhibition of proliferation, induction of apoptosis and suppression of VEGF. Based on this study and other studies, mTOR can be regarded as a potential drug target in AML.

Interleukin-13 promoter gene polymorphism -1112C/T is associated with the systemic form of mastocytosis.

BACKGROUND: Mastocytosis is a heterogenous disease involving mast cells (MC) and their progenitors. Cutaneous and systemic variants of the disease have been reported. In contrast to cutaneous mastocytosis (CM), patients with systemic mastocytosis (SM) are at risk to develop disease progression or a nonMC-lineage haematopoietic neoplasm. Little is known, however, about factors predisposing for the development of SM. One factor may be cytokine regulation of MC progenitors. METHODS: We examined the role of the interleukin-13 (IL-13) promoter gene polymorphism -1112C/T, known to be associated with increased transcription, in mastocytosis using allele-specific polymerase chain reaction method. Serum tryptase and IL-13 levels were determined by immunoassay, and expression of the IL-13 receptor in neoplastic MC by reverse transcription-polymerase chain reaction and flow cytometry. RESULTS: The frequency of the -1112T allele of the IL-13 promoter was significantly higher in patients with SM compared with CM (P < 0.008) and in mastocytosis patients compared with healthy controls (P < 0.0001). Correspondingly, the polymorphism was found to correlate with an elevated serum tryptase level (P = 0.004) and with adult-onset of the disease (P < 0.0015), both of which are almost invariably associated with SM. Serum IL-13 levels were also higher in SM patients compared with CM (P = 0.011), and higher in CT- than in CC carriers (P < 0.05). Finally, we were able to show that neoplastic human MC display IL-13 receptors and grow better in IL-13-containing medium. CONCLUSIONS: The -1112C/T IL-13 gene polymorphism and the resulting 'hypertranscription' may predispose for the development of SM.

Prognostic significance of serial determinations of lactate dehydrogenase (LDH) in the follow-up of patients with myelodysplastic syndromes.

BACKGROUND: Early recognition of disease progression in low-risk myelodysplastic syndromes (MDS) is an important decision point concerning intensive therapies. In a screen program searching for dynamic prognostic determinants, we have identified lactate dehydrogenase (LDH) as a most suitable follow-up parameter. PATIENTS AND METHODS: LDH levels were serially determined in 221 patients with de novo MDS (median age 70 years, range 24-94). The increase in LDH was correlated with survival and acute myeloid leukemia (AML) evolution. RESULTS: Confirming previous data, an elevated LDH at diagnosis was found to be associated with an increased probability of AML evolution and decreased probability of survival (P < 0.05). In the follow-up, we found that in patients who progressed (to higher IPSS category or AML), LDH levels were significantly higher in the two 3-month period preceding progression compared with the initial two 3-month period (P < 0.005). In a subgroup of patients, the increase in LDH was accompanied or followed by other signs of disease progression, such as occurrence of thrombocytopenia or appearance of circulating blasts. In multivariate analyses, the LDH increase was found to be an independent prognostic variable. CONCLUSIONS: LDH is an interesting follow-up parameter in MDS, which may assist in early recognition of disease progression and thus help in risk stratification and patient selection for interventional therapies.

Evaluation of antileukaemic effects of rapamycin in patients with imatinib-resistant chronic myeloid leukaemia.

BACKGROUND: Recent data suggest that the mammalian target of rapamycin (mTOR) is involved in the regulation of growth of neoplastic cells in chronic myeloid leukaemia (CML). PATIENTS AND METHODS: We treated six patients with imatinib-resistant CML in haematological relapse (leukocytes > 20,000 microL(-1)) with rapamycin at 2 mg per os daily for 14 consecutive days, with dose-adjustment allowed to reach a target rapamycin serum concentration of 10-20 pg mL(-1). RESULTS: A major leukocyte response with decrease to less than 10,000 microL(-1) was obtained in two patients, and a minor transient response was seen in two other patients. In responding patients, we also observed a decrease in vascular endothelial growth factor (VEGF) mRNA levels in circulating leukaemic cells. Side effects during rapamycin treatment were mild in most patients. In one patient, pneumonia developed. Rapamycin was also found to counteract growth of CML cells in vitro as determined by (3)H-thymidine incorporation. Moreover, rapamycin inhibited the growth of Ba/F3 cells exhibiting various imatinib-resistant mutants of BCR/ABL, including the T315I variant that exhibits resistance against most currently available BCR/ABL kinase inhibitors. CONCLUSIONS: Rapamycin shows antileukaemic effects in imatinib-resistant CML in vitro and in vivo. Larger trials with rapamycin or rapamycin-derivatives in combination with other targeted drugs are warranted to further determine clinical efficacy in CML.

CD203c is overexpressed on neoplastic mast cells in systemic mastocytosis and is upregulated upon IgE receptor cross-linking.

The ectoenzyme E-NPP3 (CD203c) has recently been identified as a novel activation-linked cell surface antigen on basophils. In the present study, we examined expression of CD203c on normal mast cells (MC)and bone marrow (bm) MC derived from 85 patients with systemic mastocytosis (SM), including cases with indolent SM (ISM, n=72), SM with associated clonal hematologic non-MC-lineage disease (SM-AHNMD, n=6), aggressive SM (ASM, n=3), and mast cell leukemia (MCL, n=4). Surface expression of CD203c was analyzed by multicolor flow cytometry. In patients with SM, bm MC expressed significantly higher amounts of CD203c compared to normal bm MC (median MFI in controls: 260 versus median MFI in SM: 516, p<0.05). Slightly lower amounts of CD203c were detected on MC in SM-AHNMD and ASM compared to ISM. To demonstrate CD203c expression in MC at the mRNA level, neoplastic MC were highly enriched by cell sorting, and were found to express CD203c mRNA in RT-PCR analysis. Cross-linking of the IgE receptor on MC resulted in a substantial upregulation of CD203c, whereas the KIT-ligand stem cell factor (SCF) showed no significant effects. In conclusion, CD203c is a novel activation-linked surface antigen on MC that is upregulated in response to IgE receptor cross-linking and is overexpressed on neoplastic MC in patients with SM.

Protection against woodchuck hepatitis virus (WHV) infection by gene gun coimmunization with WHV core and interleukin-12.

Woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) are closely similar with respect to genomic organization, host antiviral responses, and pathobiology of the infection. T-cell immunity against viral nucleocapsid (HBcAg or WHcAg) has been shown to play a critical role in viral clearance and protection against infection. Here we show that vaccination of healthy woodchucks by gene gun bombardment with a plasmid coding for WHcAg (pCw) stimulates proliferation of WHcAg-specific T cells but that these cells do not produce significant levels of gamma interferon (IFN-gamma) upon antigen stimulation. In addition, animals vaccinated with pCw alone were not protected against WHV inoculation. In order to induce a Th1 cytokine response, another group of woodchucks was immunized with pCw together with another plasmid coding for woodchuck interleukin-12 (IL-12). These animals exhibited WHcAg-specific T-cell proliferation with high IFN-gamma production and were protected against challenge with WHV, showing no viremia or low-level transient viremia after WHV inoculation. In conclusion, gene gun immunization with WHV core generates a non-Th1 type of response which does not protect against experimental infection. However, steering the immune response to a Th1 cytokine profile by IL-12 coadministration achieves protective immunity. These data demonstrate a crucial role of Th1 responses in the control of hepadnavirus replication and suggest new approaches to inducing protection against HBV infection.

Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility.

Protein C inhibitor (PCI) is a nonspecific, heparin-binding serpin (serine protease inhibitor) that inactivates many plasmatic and extravascular serine proteases by forming stable 1:1 complexes. Proteases inhibited by PCI include the anticoagulant activated protein C, the plasminogen activator urokinase, and the sperm protease acrosin. In humans PCI circulates as a plasma protein but is also present at high concentrations in organs of the male reproductive tract. The biological role of PCI has not been defined so far. However, the colocalization of high concentrations of PCI together with several of its target proteases in the male reproductive tract suggests a role of PCI in reproduction. We generated mice lacking PCI by homologous recombination. Here we show that PCI(-/-) mice are apparently healthy but that males of this genotype are infertile. Infertility was apparently caused by abnormal spermatogenesis due to destruction of the Sertoli cell barrier, perhaps due to unopposed proteolytic activity. The resulting sperm are malformed and are morphologically similar to abnormal sperm seen in some cases of human male infertility. This animal model might therefore be useful for analyzing the molecular bases of these human conditions.

Vascular endothelial growth factor and its receptors in male fertility.

OBJECTIVE: To determine the concentration of vascular endothelial growth factor in the seminal fluid, the presence of vascular endothelial growth factor receptors Flt-1 and KDR in spermatozoa, and the predictive value of seminal vascular endothelial growth factor on fertilization and the chance of pregnancy in patients undergoing intracytoplasmic sperm injection (ICSI) or IVF. DESIGN: Retrospective analysis. SETTING: Private institute (semen collection, IVF/ICSI) and academic research environment (analysis of seminal fluid and spermatozoa). PATIENT(S): Eighty men whose spermatozoa were subsequently used for IVF or ICSI. INTERVENTION(S): Seminal vascular endothelial growth factor was measured by an EIA. Spermatozoa were analyzed by fluorescence-activated cell sorter analysis and by immunocytochemistry. MAIN OUTCOME MEASURE(S): Oocyte fertilization rate, pregnancy rate, and presence of vascular endothelial growth factor receptors on spermatozoa. RESULT(S): Patients with a seminal concentration of vascular endothelial growth factor of 2-100 ng/mL had a sixfold increased chance of pregnancy. Vascular endothelial growth factor concentration and patient's age remained the only independent prognostic factors for pregnancy. The concentration of vascular endothelial growth factor did not correlate with indices of male factor infertility or with the oocyte fertilization rate. Expression of vascular endothelial growth factor receptors (Flt-1, KDR) on spermatozoa was demonstrated. CONCLUSION(S): The seminal concentration of vascular endothelial growth factor correlates with the chance of pregnancy in patients undergoing IVF or ICSI. Vascular endothelial growth factor receptors Flt-1 and KDR were detected on spermatozoa for the first time.

Protein C inhibitor is expressed in keratinocytes of human skin.

Protein C inhibitor is a member of the serpin family that inhibits a variety of serine proteases. Protein C inhibitor is present in numerous body fluids and is produced in the liver and by various epithelial cells. To determine if this epithelial serpin is present in skin, immunohistochemical studies were performed that showed strong staining for protein C inhibitor antigen in the epidermis. Protein C inhibitor mRNA was detected in the keratinocyte cell line HaCaT and the epidermoid carcinoma cell line A431 using reverse transcription-polymerase chain reaction suggesting that also in normal skin protein C inhibitor is derived from keratinocytes. Conditioned media from these cell lines were analyzed on immunoblots, which revealed a protein C inhibitor-antigen band that comigrated with protein C inhibitor derived from the hepatoma cell line HepG2. Using an enzyme-linked immunosorbent assay specific for total protein C inhibitor antigen the accumulation of protein C inhibitor in the cell culture supernatants of HaCaT keratinocytes was found to be 0.3 ng per h per 1 million cells. This is similar to the amount of plasminogen activator inhibitor-1 produced by these cells, which also produce tissue plasminogen activator and urokinase. Fluorescence-activated cell sorter analysis revealed similar expression of intracellular protein C inhibitor antigen in proliferating and confluent HaCaT cells. These findings demonstrate that protein C inhibitor antigen is present in the normal epidermis and that protein C inhibitor is constitutively expressed by keratinocytes in culture. Therefore, protein C inhibitor may provide protease inhibitory activity not only to internal, but also to the external surface of the body. Additionally, protein C inhibitor could contribute to the regulation of retinoid supply in the epidermis, as we have shown recently that retinoic acid binds specifically to protein C inhibitor.