Lenalidomide as immune adjuvant to a dendritic cell vaccine in chronic lymphocytic leukemia patients.

OBJECTIVES: We previously showed that immunization with ex vivo- generated autologous dendritic cells loaded with apoptotic tumor cells (Apo-DC) potentiated tumor-specific immunity in chronic lymphocytic leukemia (CLL) patients. Here, we evaluated safety and immunogenicity of Apo-DC in combination with lenalidomide, granulocyte-macrophage colony-stimulating factor (GM-CSF), and low-dose cyclophosphamide (CTX). METHODS: Ten previously untreated patients with slowly progressing CLL received 5 Apo-DC vaccinations and lenalidomide orally for 24 weeks either alone (cohort I, n = 5) or together with subcutaneous GM-CSF and intravenous CTX (cohort II, n = 5). Tumor-specific T-cell responses were measured by proliferation and IFN-γ ELISPOT assays. Immune monitoring was performed by flow cytometry. RESULTS: Dose-limiting toxicity was observed in 3/10 patients, 2 in cohort I and one in cohort II. One patient developed autoimmune hemolytic anemia and another grade 4 thrombocytopenia. Vaccine-induced immune responses were seen in 5/5 and 4/5 patients in cohort I and II, respectively. The expression of immune checkpoints on T cells did not change significantly. CONCLUSIONS: Lenalidomide alone or in combination with GM-CSF and low-dose CTX as immune adjuvant to the Apo-DC vaccine elicited tumor-specific T-cell responses in CLL patients. However, unexpected toxicity was observed and caution is suggested in further exploring this drug as immune adjuvant in CLL.

The Barrow Neurological Institute screen for higher cerebral functions in cognitive screening after stroke.

The objective of this study was to evaluate the Barrow Neurological Institute Screen for Higher Cerebral Functions (BNIS) in screening for cognitive dysfunction at long-term follow-up after stroke in young and middle-aged patients. Within the Sahlgrenska Academy Study on Ischemic Stroke Outcome, the BNIS and the Mini-Mental State Examination (MMSE) were administered to 295 consecutive surviving patients seven years after ischemic stroke. All participants were less than 70 years at index stroke. BNIS score less than 47 and an MMSE score less than 29 were chosen to indicate cognitive dysfunction. Two hundred eighty-one (95%) patients completed both tests. The 2 test scores were moderately correlated, and both tests correlated to disability as measured by the modified Rankin Scale. The distribution of the MMSE score was skewed toward the top scores, with a marked ceiling effect, whereas the BNIS score was more normally distributed. Most BNIS subscales showed mean performance around the mid of the scale without ceiling effects. Both tests identified a large proportion of the subjects as cognitive impaired, however, with a substantially larger proportion for the BNIS (89%) compared with the MMSE (65%). We conclude that the BNIS may be a useful screening instrument for cognitive dysfunction after ischemic stroke and that a large proportion of young and middle-aged ischemic stroke survivors showed signs of cognitive dysfunction long after index stroke. Further validations of BNIS against formal neuropsychological testing and studies of the determinants and consequences of long-term cognitive outcome in this patient group are warranted.

Killing of CLL and NHL cells by rituximab and ofatumumab under limited availability of complement.

Rituximab and ofatumumab are anti-CD20 antibodies applicable to treatment for non-Hodgkin's lymphoma and chronic lymphocytic leukemia (CLL). Effectiveness of both immunotherapeutics may depend on exhaustible complement system. To model the efficacy of complement usage by ofatumumab and rituximab under limited complement availability, we compared complement-dependent cytotoxicity exerted by these antibodies at low (5 and 10%) and physiological (50%) serum concentration in twelve CD20-positive cell lines and six freshly isolated CLL cells. Simultaneously, we assessed the expression of CD20 and membrane-bound complement inhibitors. Ratios of CD20 to CD59 and/or CD55 distinguished highly sensitive cells lysed equally efficient by both antibodies from the moderately sensitive cells, which were killed more efficiently by ofatumumab.

Vaccination with dendritic cells loaded with tumor apoptotic bodies (Apo-DC) in patients with chronic lymphocytic leukemia: effects of various adjuvants and definition of immune response criteria.

We previously demonstrated that autologous dendritic cells that have endocytosed apoptotic bodies of chronic lymphocytic leukemia (CLL) cells (Apo-DC) can stimulate antileukemic T cell responses in vitro. In this phase I study, we vaccinated 15 asymptomatic CLL patients at five time points with Apo-DC administered intradermally either alone (cohort I), or in combination with subcutaneous granulocyte-macrophage-colony-stimulating-factor (GM-CSF) (cohort II) or with GM-CSF and intravenous low-dose cyclophosphamide (cohort III). Aim of the study was to evaluate the safety and immunogenicity of Apo-DC alone or in combination with GM-CSF and low-dose cyclophosphamide in CLL patients. All patients completed the vaccination schedule without dose-limiting toxicity. No objective clinical responses were seen. Vaccine-induced leukemia-specific immune responses were evaluated by IFN-γ ELISpot and proliferation assays over a 52 weeks observation period and immune response criteria were defined. According to these criteria, 10/15 patients were defined as immune responders. The frequency of immune-responding patients was higher in cohorts II (3/5) and III (5/5) than in cohort I (2/5). In order to further characterize the induced immune response, estimation of secreted cytokines and CD107-degranulation assay were performed. Clustering of T and CLL cells was observed in CD107-degranulation assay and visualized by confocal microscopy. Additionally, assessment of regulatory T cells (T(regs)) revealed their significantly lower frequencies in immune responders versus non-responders (P < 0.0001). Cyclophosphamide did not reduce T(regs) frequency. In conclusion, vaccination with Apo-DC + GM-CSF and cyclophosphamide was safe and elicited anti-CLL immune responses that correlated inversely with T(regs) levels. Lack of clinical responses highlights the necessity to develop more potent vaccine strategies in B cell malignancies.

Long-term effects of idiotype vaccination on the specific T-cell response in peripheral blood and bone marrow of multiple myeloma patients.

OBJECTIVES: To elucidate long-term effects of idiotype (Id) vaccination on Id-specific T cells of multiple myeloma (MM) patients and compare Id-specific T-cell responses of peripheral blood with those of bone marrow (BM). MATERIALS AND METHODS: Id-specific T-cell responses of peripheral blood mononuclear cells (PBMC) were compared with those of BM mononuclear cells (BMMC) in 10 MM patients vaccinated with the Id protein at a median time of 41 months since the last immunization. The PBMC responses at late follow-up were also compared with those during active immunization. The responses were assessed by a proliferation assay, enzyme-linked immunospot (ELISPOT) (gamma-interferon), cytometric bead array (CBA) for secreted cytokines and quantitative real-time polymerase chain reaction (QRT-PCR) for cytokine gene expression. RESULTS: At the late testing time, an Id-specific response was detected in PBMC of five patients (ELISPOT, CBA, QRT-PCR) and in BMMC of four patients (CBA, QRT-PCR). A response in both compartments was noted only in three patients. The cytokines gene profile was consistent with a predominance of Th(2) cells [interleukin (IL)-4, IL-5, IL-10]. Comparison of the Id-specific responses of PBMC during active immunization with those at the late follow-up showed that the frequency and magnitude of the responses had decreased significantly by time (proliferation/ELISPOT) (P < 0.02) and shifted at the gene level from a Th(1) to a Th(2) profile (P < 0.05). CONCLUSION: Id-specific T-cells may decline overtime and shift toward a Th(2) response and may be found at a similar frequency of patients in blood and BM.

Idiotype protein vaccination in combination with adjuvant cytokines in patients with multiple myeloma--evaluation of T-cell responses by different read-out systems.

Anti-idiotypic T cells were analyzed in myeloma patients (n=18) vaccinated with idiotypic protein together with the adjuvant cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-12 (IL-12). In the group given IL-12/GM-CSF, 78% developed idiotype specific T cells as compared to 22% in the group given only IL-12 (proliferation/ELISPOT assays) (p<0.05). The percentage of immune-responding patients increased when quantitative real time polymerase chain reaction assays for cytokines were included. A predominance of a Th1 (IFN-gamma/TNF-alpha) immune response was noted in the IL-12 group while a Th2 (IL-5) response prevailed in the IL-12/GM-CSF group (p=0.053). Application of multiple read-out systems improved the characterization of the immune response.

Age-correlated decline in [3H]tiagabine binding to GAT-1 in human frontal cortex.

BACKGROUND AND AIMS: In spite of the fact that GABA is a significant transmitter, little is known about the GABA system in aging, compared with other transmitter systems. [(3)H]tiagabine is a ligand for GABAergic neurons, which binds with 10-fold higher affinity to the GABA uptake site than [(3)H]nipecotic acid. The aim of this study was to study the binding of [(3)H]tiagabine to the GABA transporter 1, GAT-1, in human frontal cortex and cingulate cortex from individuals of varying ages. METHODS: [(3)H]tiagabine binding experiments were conducted on post-mortem brain tissue from 19 individuals (age range 17-78 years) without known neurological or psychiatric disorders. Binding data vs age and postmortem interval was analysed by Pearson correlation. RESULTS: The density of [(3)H]tiagabine binding to GAT- 1 decreased significantly with increasing age in the frontal cortex, whereas binding affinity was unchanged. No significant alterations in binding parameters were observed in the cingulate cortex. No correlation was found between post-mortem delay and the number of [(3)H]tiagabine binding sites. CONCLUSIONS: According to the present study, presynaptical alterations in the GABA system are correlated with aging in the frontal cortex of the human brain. Further studies involving a broader range of brain regions seem warranted, to confirm the present findings and to enlarge knowledge about the GABA system in aging.

SALSA: a simulation tool to assess ecological sustainability of agricultural production.

In order to assess the ecological sustainability of agricultural production systems, there is a need for effective tools. We describe an environmental systems analysis tool called SALSA (Systems Ana/ysis for Sustainable Agriculture). It consists of substance/material flow models in which the simulation results are interpreted with life-cycle assessment methodology. The application of SALSA is demonstrated in a case study in which three different ways of producing pigs are compared with respect to energy input and the environmental impacts of global warming, eutrophication, and acidification. The scenario that combined a low-protein diet without soy meal with an improved manure-management technique with low nitrogen losses was the best for all impact categories studied. The strength of the SALSA models was their capacity to capture consequences of management options that had an influence on several processes on a farm, which enabled the type of complex studies we describe.

[(3)H]Tiagabine binding to GABA transporter-1 (GAT-1) in suicidal depression.

BACKGROUND: In depressive disorders, alterations of GABA concentrations and number of postsynaptic GABA related receptor binding sites, as well as antidepressant effects exerted by GABA agonists, suggest a pathogenetic involvement of the GABA system. METHOD: The binding of the presynaptic GABA ligand [(3)H]tiagabine to GABA transporter-1 (GAT-1) was studied in post mortem human frontal cortex and cingulate gyrus from 13 suicide victims and 19 controls without known neurological or psychiatric disorder. RESULTS: No differences were found between the suicide victims and the controls with regard to the number of [(3)H]tiagabine binding sites (B(max)) or apparent affinity (K(d)). LIMITATIONS: The study was limited to two brain regions. CONCLUSION: Findings in other studies of alterations in the GABA system in depression seem according to the present results not to be associated with significant changes in the GABA uptake binding sites in the regions investigated.

Increased [(3)H]tiagabine binding to GAT-1 in the cingulate cortex in schizophrenia.

Postmortem samples from individuals with schizophrenia (n = 13) and control subjects (n = 10) were investigated for binding of [(3)H]tiagabine to GABA transporter-1 GAT-1. The binding was analyzed in the cingulate cortex and the caudate nucleus. There were no differences in binding affinity between the groups in any of the investigated areas. The maximum number of binding sites (B(max)) was elevated in the schizophrenic cingulate cortex compared to controls (1,264 +/- 96 vs. 860 +/- 123 fmol/mg of protein). The B(max) in the caudate nucleus for schizophrenics (426 +/- 40 fmol/mg of protein) was the same as for controls (495 +/- 69 fmol/mg of protein). The increase in GAT-1 in schizophrenia could be explained by a modulatory upregulation in the cingulate cortex.