Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies.

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

Prognostic relevance of topoisomerase II α and minichromosome maintenance protein 6 expression in colorectal cancer.

BACKGROUND: Despite rising incidence rates of colorectal malignancies, only a few prognostic tools have been implemented in proven clinical routine. Cell division and proliferation play a significant role in malignancies. In terms of colorectal cancer, the impact of proliferation associated proteins is controversially debated. The aim of our study was to examine the expression of topoisomerase II α and minichromosome maintenance protein 6 and to correlate these findings with the clinical data. METHODS: Tissue samples of 619 patients in total were stained using the antibodies Ki-S4 and Ki-MCM6 targeting topoisomerase II α as well as minichromosome maintenance protein 6. The median rate of proliferation was correlated with clinical and follow up data. RESULTS: The expression rate of minichromosome maintenance protein 6 is significantly higher than the proportion of topoisomerase II α in tumour cells (p < 0.001). A high expression of both proteins coincides with a beneficial outcome for the patient, indicating a favourable prognostic marker (p < 0.001 and p = 0.008). CONCLUSIONS: We have demonstrated that high expression rates of proliferative markers is linked to a beneficial patient outcome. According to the general opinion, a high expression rate correlates with a poor patient outcome. In this study, we were able to refute this assertion.

Suppression of MAP kinases inhibits microglial activation and attenuates neuronal cell death induced by alpha-synuclein protofibrils.

Alpha-Synuclein (alpha-Syn) accounts, as a major component of Lewy bodies (LB), for the filamentous deposits in many cases of neurodegenerative diseases. Yet, little is known about the molecular mechanisms of neuronal loss in these diseases. The correlation between alpha-Syn oligomerization/aggregation and pathologies raises the key question of which molecular form of alpha-Syn (i.e. monomeric alpha-Syn, protofibrils or mature fibrils) represents the damage-inducing culprit in the scenario of synucleinopathies. We show that human alpha-Syn protofibrils (PFs) are potent activators of parallel proinflammatory signalling pathways (p38 and ERK1/2 MAP kinases and NF-kappaB) in microglial cells in vitro. Furthermore, stereotactic injection of alpha-Syn PFs into the substantia nigra of adult rats leads to a profound activation of microglia and adjacent neuronal cell loss, which can be attenuated by the MAP kinase inhibitor semapimod. We propose that the neurodegenerative process of alpha-synucleinopathies involves microglial activation through alpha-Syn released or extruded from cells with pathogenic alpha-Syn metabolism. Compounds that inhibit the MAPK/NF-kappaB pathways might be a promising pharmacological strategy for the treatment of the inflammatory component of synucleinopathies including PD.

Involvement of benzodiazepine receptors in neuroinflammatory and neurodegenerative diseases: evidence from activated microglial cells in vitro.

Increased binding of a ligand for the peripheral benzodiazepine binding receptor is currently used in PET studies as an in vivo measurement of inflammation in diseases like multiple sclerosis and Alzheimer's disease. Although peripheral-type benzodiazepin receptors (PBRs) are abundant in many cell types and expressed in the CNS physiologically only at low levels, previous reports suggest that after experimental lesions in animal models and in human neurodegenerative/-inflammatory diseases upregulated PBR expression with increased binding of its ligand PK11195 is confined mainly to activated microglia in vivo/in situ. Because the functional role of the PBR is unknown, we confirm by immunohistochemistry and PCR (I) that this receptor is expressed on microglia in vitro and (II) that benzodiazepines modulate proliferation of microglial cells and the release of the inflammatory molecules nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) in cell culture supernatants of primary rat microglia. Compared to lipopolysaccharide-activated controls the release of NO was markedly decreased in cultures treated with benzodiazepines (clonazepam, midazolam, diazepam) and the PBR ligand PK11195. Moreover, release of TNF-alpha and proliferation was significantly inhibited in the benzodiazepine-treated groups. These findings link the in vivo data of elevated PBR levels in neurodegenerative/-inflammatory diseases to a functional role and opens up possible therapeutic intervention targeting the PBR in microglia.

The efficacy of two bupivacaine hydrochloride injection products.

OBJECTIVE: The relative efficacy of two bupivacaine hydrochloride injection products was investigated in patients who were undergoing intra-ocular eye surgery. DESIGN: Patients took part in this double-blind, randomised, parallel-group study and received either Macaine (Keatings) or Regibloc (Intramed), according to the randomisation schedule. SETTING: The study was carried out in the ophthalmology operating theatres of National and Pelonomi Hospitals, Bloemfontein, South Africa. PATIENTS: Thirty male and 74 female patients who needed extra-capsular lens extraction plus intra-ocular lens implantation, extra-capsular lens extraction, or trabeculectomy were selected for the study. OUTCOME MEASURES: Akinesia was evaluated after 10, 15 and 20 minutes. In the event of incomplete akinesia after 20 minutes, an additional injection was administered, and after 5 minutes another evaluation of akinesia was done. Anaesthesia was evaluated at the beginning of surgery. RESULTS: The proportions of patients who received no additional anaesthesia were 57.7% for Macaine and 70.8% for Regibloc (difference 13.1%, 95% confidence interval (CI) -5.5 - 31.7%). The proportions of patients with adequate akinesia (possibly after additional anaesthesia) were 90.4% for Macaine and 89.6% for Regibloc (difference -0.8%, 95% CI-12.6 - 11.0%). The proportions of patients experiencing no pain or discomfort at the beginning of surgery were 88.2% for Macaine and 87.5% for Reglibloc (difference -0.7%, 95% CI-13.6 - 12.1%). CONCLUSION: The study results indicate that Regibloc is at least as effective as, or superior to, Macaine in achieving adequate akinesia.

Mononuclear cells from patients with the hyper-IgE syndrome produce little IgE when they are stimulated with recombinant human interleukin-4.

To investigate whether B cells from patients with the hyper-IgE syndrome are more sensitive to the effects of interleukin-4 in vitro than B cells of normal or atopic individuals, we stimulated blood mononuclear cells (MNC) with varying doses of recombinant human interleukin 4 (rhIL-4) and measured supernatant IgE concentrations after 18 days of culture. Geometric mean spontaneous IgE synthesis after 18 days of culture without rhIL-4 was low (less than 3 ng/ml) and similar for MNCs from nine patients with the hyper-IgE syndrome, nine atopic and nine normal subjects. As found in our previous studies, MNCs from the nine atopic and the nine normal donors produced significant and similar quantities of IgE (geometric mean maximum IgE, 25.2 and 18.7 ng/ml, respectively) when MNCs were stimulated with rhIL-4. MNCs from both donor groups had similar sensitivity to the concentration of IL-4 eliciting the IgE response. In striking contrast, MNCs from the nine patients with the hyper-IgE syndrome failed to produce significant IgE over that produced spontaneously when MNCs were stimulated by a wide range of rhIL-4 concentrations. Coculture of B cell-enriched subpopulations from patients with the hyper-IgE syndrome with T cell-enriched subpopulations from nonatopic and atopic donors failed to restore responsiveness to rhIL-4. The addition of anti-CD40 monoclonal antibody to MNC cultures did result in enhancement of rhIL-4 IgE synthesis by MNCs from patients with the hyper-IgE syndrome, but the concentration of anti-CD40 required to elicit this enhancement was tenfold higher than for control MNCs.(ABSTRACT TRUNCATED AT 250 WORDS)