Establishment of Cytotoxic and Non-cytotoxic NK-92 Cell Clones - Evaluation of Their Function as Target Cells.

BACKGROUND/AIM: Previous studies have demonstrated that NK cells present in PBMCs might explain why clinical trials conducted with NK-92 as well as CAR modified NK-92 cells have to a large extent failed. Two NK-92 clones with different NK target cell properties have been established and are described here. MATERIALS AND METHODS: Two NK-92 cell clones, NK-92 clone 1 and clone 2, were established using the limiting dilution technique. A time-resolved fluorometric assay (TDA-labeled NK-92 clone 1, 2 or K562 as target cells) was used for measuring their sensitivities to NK cell-mediated cytolysis and their NKG2D expression was identified with immunoblotting. RESULTS: A striking difference between the NK-92 clones in their cytotoxic capacity against K562 cells was observed. A clear correlation was noticed between these NK-92 clones when used as target cells and their ability to kill K562 cells. A 50:1 effector:target ratio (PBMCs:NK-92 clone 1) gave 6.50±5.44% lysis whereas the corresponding value was 39.9±10.0% with NK-92 clone 2 as target cells. Interestingly, incubating PBMCs in medium for longer times slightly potentiated their NK activity also against the NK-92 clone 1 (E:T ratio 50:1), from 2.5±0.88% lysis (24 h pre-incubation time) to 13.7±9.04% (48 h) and 13.8±6.89% (72 h). Immunoblotting with anti-NKG2D antibodies stained an approximately 34 kDa protein band in lysates prepared from NK-92 clone 1 cells, which corresponds to the NKG2D antigen. A very faint band of the same size was observed in lysates prepared from NK-92 clone 2 cells. CONCLUSION: The NK-92 clones 1 and 2, established and described here, might turn out to be very useful for finding possible solutions for using NK-92 and CAR NK-92 cells in future treatments of human malignant diseases.

Human IL-15 Inhibits NK Cells Specific for Human NK-92 Cells.

BACKGROUND/AIM: Recent studies have indicated that natural killer (NK) cells present in peripheral blood mononuclear cells (PBMCs) might be responsible for the somewhat poor outcome of clinical trials conducted with the NK cell line NK-92, as well as chimeric antigen receptor-modified NK-92 cells against leukemias and lymphomas. These NK cells and how their cytotoxic profiles can be altered by some common gamma chain receptor-dependent cytokines or by removal of CD4+ cells have been addressed herein. MATERIALS AND METHODS: A time-resolved fluorometric assay using 2.2':6'.2"-terpyridine-6.6"-dicarboxylic acid-labeled NK-92 or K562 as target cells was used for measuring the cytotoxic activity of cytokine-treated PBMCs and purified NK cells. RESULTS: Pre-incubation with 25 ng/ml interleukin 12 (IL-12), IL-15 or IL-21 for 72 h increased NK cell activity against K562 cells by more than 90% (1:25 target:effector ratio), whereas the corresponding NK cell activity against NK-92 cells was reduced by 15.9±0.1% by IL-12 and 50.6±2.9% by IL-15 compared to cells treated with medium alone. IL-7, on the other hand, increased NK activity against K562 to a much smaller extent (10.4±0.4%) and inhibited NK-92 cell lysis by 15.2±0.3%. Interestingly, similar amounts of IL-2 potentiated NK cell activity against both K562 and NK-92 cells by 50.9±0.5% and 14.3±0.9%, respectively. Purification of NK cells with magnetic beads demonstrated that NK cells indeed were responsible for the observed cytotoxic activity against both NK-92 cells (58.5±9.10%, 1:100 target:effector ratio) and K562 cells (81.6±9.57%, 1:100 target:effector ratio). Elimination of CD4+ cells from PBMCs did not alter the NK activity profile. CONCLUSION: This study highlights a problem that might arise with immune-based NK-92 and chimeric antigen receptor-transduced NK-92 cell therapies and pinpoints the need for evaluating new NK-like cell lines.

Human NK-92 Cells Function as Target Cells for Human NK Cells - Implications for CAR NK-92 Therapies.

BACKGROUND/AIM: Recent studies indicate that chimeric antigen receptor (CAR)-T-cells seem to be superior to CAR modified NK-92 cells. One, at least partial, explanation to this discrepancy has been addressed herein, by having NK-92 cells as target cells in cytotoxicity reactions using peripheral blood mononuclear cells. MATERIALS AND METHODS: A time-resolved fluorometric assay (TDA-labeled NK-92 or K562 as target cells) was used for measuring the cytotoxic activity of blood mononuclear cells (PBMC). RESULTS: The cytotoxic capacity of the NK-92 cells was initially demonstrated by their ability to efficiently kill K562 cells. Interestingly, having PBMC as effector cells rendered the very same NK-92 cells sensitive to NK-cell mediated cytolysis. A 1:100 target:effector ratio gave 34.1% lysis compared to 72.2% lysis for K562 cells. Incubating PBMC for longer times (24 up to 48 h) potentiated their NK-activity against NK-92 cells even more, reaching a level close to that obtained with K562 cells. CONCLUSION: This study pinpoints a severe problem that has to be considered in future immune-based cancer therapies with NK-92 as well as CAR-transduced NK-92 cells.

Evaluation of the Drug Use Disorders Identification Test (DUDIT) in criminal justice and detoxification settings and in a Swedish population sample.

Psychometric properties of the 11-item self-report Drug Use Disorders Identification Test (DUDIT) were evaluated in a sample of heavy drug users from prison, probation, and inpatient detoxification settings, and in a general Swedish population sample. In the drug user sample, the DUDIT predicted drug dependence with a sensitivity of 90% for both DSM-4 and ICD-10 and a respective specificity of 78 and 88%. Reliability according to Cronbach's alpha coefficient was 0.80. In the population sample, 3.1% scored positive on the DUDIT; T-score values are suggested. The DUDIT screens effectively for drug-related problems in clinically selected groups and may prove useful in the context of public health surveys.

Fetus at risk: prevalence of alcohol consumption during pregnancy estimated with a simple screening method in Swedish antenatal clinics.

AIMS: Maternal alcohol consumption is a major health hazard for the fetus. Sweden has an extensive system of public antenatal care clinics, whose mission is to detect and prevent this type of health hazards. However, very few cases of alcohol consumption during pregnancy are detected. The aim of this study was to examine the prevalence of hazardous or harmful alcohol consumption during pregnancy in a consecutive series of Swedish pregnant females. DESIGN, SETTING, PARTICIPANTS AND MEASUREMENTS: The Alcohol Use Disorders Identification Test (AUDIT) was used to collect anonymous data from consecutive pregnant subjects admitted during 1 year to an antenatal clinic in Stockholm, and signing up for parental education offered routinely (n = 1327). Data were obtained from 1101 subjects, typically in pregnancy week 30. A complete AUDIT form was filled out referring to alcohol use during the year prior to pregnancy. A separate form with the consumption items from AUDIT was filled out to report behaviour during pregnancy. FINDINGS: For the year preceding pregnancy, 17% of subjects reported AUDIT scores of 6 or higher, indicating hazardous or harmful alcohol use in women. Few individuals reported scores of 13 or higher (indicating abuse or dependence), but almost half the subjects (46%) reported binge drinking (six standard drinks on a single occasion) once/month or more often, and 6% reported binge drinking on every occasion of alcohol consumption. One-third of the subjects (30%) continued regular alcohol use during pregnancy, and 6% reported consumption two to four times/month. In a logistic regression model, AUDIT scores for the year prior to pregnancy and subject age, but not education level were significant predictors of continued alcohol use during pregnancy. CONCLUSIONS: Alcohol use during pregnancy is more extensive than has been presumed in Sweden. Simple, clinically useful screening methodology detects hazardous consumption during pregnancy in a manner which regular antenatal care does not. If this methodology can be shown to have similar sensitivity when administered under non-anonymous conditions, it should be made part of routine antenatal care.

Two NOTCH4 polymorphisms and their relation to schizophrenia susceptibility and different personality traits.

BACKGROUND: Recently, linkage disequilibrium mapping of the major histocompatibility complex region on the short arm of human chromosome 6 suggested that the NOTCH4 locus is highly associated with schizophrenia. OBJECTIVES AND METHODS: We analysed two polymorphisms in this gene in Swedish schizophrenic patients ( =74) and control subjects ( =135). The NOTCH4 variants were also analysed in schizophrenic patients with regard to subdiagnosis, age at first hospitalization, abuse/dependence of alcohol, solvents, or drugs, previous suicide attempts, extrapyramidal symptoms, treatment with anticholinergic drugs, and response to anti-psychotic drug treatment. Control subjects were scrutinized with regard to personality, another partially heritable trait suggested being of importance in schizophrenia. In addition, two intermediate endophenotypes suggested being of importance in schizophrenia, dopamine D(2) receptor density in striatum and monoamine metabolites in cerebrospinal fluid, respectively, were investigated with regard to the two NOTCH4 variants. RESULTS: There was no significant association between the patients and the controls for the two investigated polymorphisms neither for the parameters analysed in the schizophrenia material. The NOTCH4 SNP2 variant, an A-->G substitution, was associated with the Karolinska Scales of Personality Irritability scale. The NOTCH4 (CTG)(n) variant was associated with the revised NEO personality inventory Extraversion and Activity (E4) scales. However, after correction for multiple testing, no difference remained significant. The results for the endophenotypes and the polymorphisms were non-significant. CONCLUSIONS: The present study does not support that the investigated NOTCH4 variants have a major influence on susceptibility to schizophrenia or related neurobiological traits.