Functional assessment for clinical use of serum-free adapted NK-92 cells

Natural killer (NK) cells stand out as promising candidates for cellular immunotherapy due to their capacity to kill malignant cells. However, the therapeutic use of NK cells is often dependent on cell expansion and activation with considerable amounts of serum and exogenous cytokines. We aimed to develop an expansion protocol for NK-92 cells in an effort to generate a cost-efficient, xeno-free, clinical grade manufactured master cell line for therapeutic applications. By making functional assays with NK-92 cells cultured under serum-free conditions (NK-92(SF)) and comparing to serum-supplemented NK-92 cells (NK-92(S)) we did not observe significant alterations in the viability, proliferation, receptor expression levels, or in perforin and granzyme levels. Interestingly, even though NK-92(SF) cells displayed decreased degranulation and cytotoxicity against tumor cells in vitro, the degranulation capacity was recovered after overnight incubation with 20% serum in the medium. Moreover, lentiviral vector-based genetic modification efficiency of NK-92(SF) cells was comparable with NK-92(S) cells. The application of similar strategies can be useful in reducing the costs of manufacturing cells for clinical use and can help us understand and implement strategies towards chemically defined expansion and genetic modification protocols

Deletion of chromosomal region 8p21 confers resistance to Bortezomib and is associated with upregulated Decoy trail receptor expression in patients with multiple myeloma

Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma (MM) that undergo autologous stem cell transplantation (ASCT). In this study, we aimed to identify the immunological and molecular consequences of del(8)(p21) with regards to treatment response and bortezomib resistance. In patients receiving bortezomib as a single first line agent without any high-dose therapy, we have observed that patients with del(8)(p21) responded poorly to bortezomib with 50% showing no response while patients without the deletion had a response rate of 90%. In vitro analysis revealed a higher resistance to bortezomib possibly due to an altered gene expression profile caused by del(8)(p21) including genes such as TRAIL-R4, CCDC25, RHOBTB2, PTK2B, SCARA3, MYC, BCL2 and TP53. Furthermore, while bortezomib sensitized MM cells without del(8)(p21) to TRAIL/APO2L mediated apoptosis, in cells with del(8)(p21) bortezomib failed to upregulate the pro-apoptotic death receptors TRAIL-R1 and TRAIL-R2 which are located on the 8p21 region. Also expressing higher levels of the decoy death receptor TRAIL-R4, these cells were largely resistant to TRAIL/APO2L mediated apoptosis. Corroborating the clinical outcome of the patients, our data provides a potential explanation regarding the poor response of MM patients with del(8)(p21) to bortezomib treatment. Furthermore, our clinical analysis suggests that including immunomodulatory agents such as Lenalidomide in the treatment regimen may help to overcome this negative effect, providing an alternative consideration in treatment planning of MM patients with del(8)(p21)

Maximizing output from non-target screening

The purpose of this project is to dig deeper into the data material already generated in the Suspect screening in Nordic countries: Point sources in city areas (TemaNord: 2017:561) to further optimize the benefits of the major work that has already been done. Samples (effluent, sediment, and biota) from all of the Nordic countries were carefully selected, sampled and analysed by a consortium of some of the Nordic region’s most experienced scientific groups in analyses of emerging environmental contaminants. But where perhaps the full potential of the generated data is still to be realized. This project will try to further identify and describe the substances already detected, to be able to better understand what substances we in modern Nordic societies release into the sea via our wastewater.

Quantitative RT-PCR analysis of gene expression in MM patients with or without del(8)(p21).

<p>TLDA cards were used to analyze mRNA levels in patients with (n = 19) and without (n = 6) del(8)(p21). For the analysis, GAPDH and ACTB genes were used as endogenous controls while RNA samples from K562 and U266 cell lines were used as calibrators. (*p<0.05, unpaired t test) (A) The mRNA level relative expression of TRAIL receptors in patients with and without the deletion. TRAIL-R1,-R2 and–R3 did not show any change while TRAIL-R4 was expressed at significantly higher levels in patients carrying the deletion. (B) Genes on or near 8p21 <b>(C)</b> other analyzed genes located elsewhere in the chromosome that show at least two-fold differential expression in MM cells with the deletion. Depicted are the gene expression levels in MM cells carrying the deletion, normalized to samples without the deletion.</p

Multivariate analysis of prognostic factors in multiple myeloma patients.

<p>Multivariate analysis of prognostic factors in multiple myeloma patients.</p

Multiple myeloma patients with del(8)(p21) have poor TTP and OS.

<p>Time to progression (TTP) of (A) all patients, (B) HDT patients, (C) non-HDT patients after diagnosis (months). Overall survival (OS) of (D) all patients, (E) HDT patients, (F) non-HDT patients after diagnosis (months). Patients with del(8)(p21) (n = 24). Patients without del(8)(p21) (n = 64). HDT patients with del(8)(p21) (n = 14). HDT patients without del(8)(p21) (n = 41). Non-HDT patients with del(8)(p21) (n = 10). Non-HDT patients without del(8)(p21) (n = 23). Log-rank (Mantel-cox) test performed using Graphpad Prism software.</p

Patients with 8p21 deletion respond poorly to bortezomib treatment.

<p>Patients diagnosed with MM and receiving bortezomib based treatment at 1^st line were selected to this study (n = 88). (<b>A)</b> left panel is the non-HDT patients that received bortezomib (Vel: Velcade®, n = 33) as 1^st line treatment and right panel is the non-HDT patients that did not respond to 1^st line bortezomib treatment or relapsed and treated with Lenalidomide based regimen as a 2^nd line treatment (n = 16). B. Left panel displays patients received high dose treatment and bortezomib as 1^st line treatment (n = 55) and right panel is the response to Lenalidomide based treatment as 2^nd line for relapse patients (n = 18). Details of the treatments are shown in materials & methods section. Chi-square test was performed to analyze results.</p

Patients with del(8)(p21) fail to upregulate pro-apoptotic TRAIL receptor expression upon bortezomib treatment.

<p>Relative expression of TRAIL receptors of 5 MM patients with del(8)(p21) and 7 MM patients without deletion is determined by flow cytometry. Cell surface Expression levels are normalized to corresponding isotype controls. <b>(A)</b> TRAIL-R1, <b>(B)</b> TRAIL-R2, <b>(C)</b> TRAIL-R3, <b>(D)</b> TRAIL-R4. Effect of bortezomib treatment on TRAIL receptor expression is analyzed by paired t-test.</p

Donepezil in Alzheimer's disease: what to expect after 3 years of treatment in a routine clinical setting.

Background/Aims: Clinical short-term trails have shown positive effects of donepezil treatment in patients with Alzheimer's disease. The outcome of continuous long-term treatment in the routine clinical settings remains to be investigated. Methods: The Swedish Alzheimer Treatment Study (SATS) is a descriptive, prospective, longitudinal, multicentre study. Four hundred and thirty-five outpatients with the clinical diagnosis of Alzheimer's disease, received treatment with donepezil. Patients were assessed with Mini-Mental State Examination (MMSE), Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog), global rating (CIBIC) and Instrumental Activities of Daily Living (IADL) at baseline and every 6 months for a total period of 3 years. Results: The mean MMSE change from baseline was positive for more than 6 months and in subgroups of patients for 12 months. After 3 years of treatment the mean change from baseline in MMSE-score was 3.8 points (95% CI, 3.0-4.7) and the ADAS-cog rise was 8.2 points (95% CI, 6.4-10.1). This is better than expected in untreated historical cohorts, and better than the ADAS-cog rise calculated by the Stern equation (15.6 points; 95% CI, 14.5-16.6). After 3 years with 38% of the patients remaining, 30% of the them were unchanged or improved in the global assessment. Conclusion: Three-year donepezil treatment showed a positive global and cognitive outcome in the routine clinical setting