Phase Ib open-label, multicenter study of pixatimod, an activator of TLR9, in combination with nivolumab in subjects with microsatellite-stable metastatic colorectal cancer, metastatic pancreatic ductal adenocarcinoma and other solid tumors.

BACKGROUND: Pixatimod is a unique activator of the Toll-like Receptor 9 pathway. This phase I trial evaluated safety, efficacy and pharmacodynamics of pixatimod and PD-1 inhibitor nivolumab in immunologically cold cancers. METHODS: 3+3 dose escalation with microsatellite stable metastatic colorectal cancer (MSS mCRC) and metastatic pancreatic ductal adenocarcinoma (mPDAC) expansion cohorts. Participants received pixatimod once weekly as a 1-hour intravenous infusion plus nivolumab every 2 weeks. Objectives included assessment of safety, antitumor activity, pharmacodynamics, and pharmacokinetic profile. RESULTS: Fifty-eight participants started treatment. The maximum tolerated dose of pixatimod was 25 mg in combination with 240 mg nivolumab, which was used in the expansion phases of the study. Twenty-one grade 3-5 treatment-related adverse events were reported in 12 participants (21%); one participant receiving 50 mg pixatimod/nivolumab had a treatment-related grade 5 AE. The grade 3/4 rate in the MSS mCRC cohort (n=33) was 12%. There were no responders in the mPDAC cohort (n=18). In the MSS mCRC cohort, 25 participants were evaluable (initial postbaseline assessment scans >6 weeks); of these, three participants had confirmed partial responses (PR) and eight had stable disease (SD) for at least 9 weeks. Clinical benefit (PR+SD) was associated with lower Pan-Immune-Inflammation Value and plasma IL-6 but increased IP-10 and IP-10/IL-8 ratio. In an MSS mCRC participant with PR as best response, increased infiltration of T cells, dendritic cells, and to a lesser extent NK cells, were evident 5 weeks post-treatment. CONCLUSIONS: Pixatimod is well tolerated at 25 mg in combination with nivolumab. The efficacy signal and pharmacodynamic changes in MSS mCRC warrants further investigation. TRIAL REGISTRATION NUMBER: NCT05061017.

Dead Cert: Measuring Cell Death.

Many cells in the body die at specific times to facilitate healthy development or because they have become old, damaged, or infected. Defects in cells that result in their inappropriate survival or untimely death can negatively impact development or contribute to a variety of human pathologies, including cancer, AIDS, autoimmune disorders, and chronic infection. Cell death may also occur following exposure to environmental toxins or cytotoxic chemicals. Although this is often harmful, it can be beneficial in some cases, such as in the treatment of cancer. The ability to objectively measure cell death in a laboratory setting is therefore essential to understanding and investigating the causes and treatments of many human diseases and disorders. Often, it is sufficient to know the extent of cell death in a sample; however, the mechanism of death may also have implications for disease progression, treatment, and the outcomes of experimental investigations. There are a myriad of assays available for measuring the known forms of cell death, including apoptosis, necrosis, autophagy, necroptosis, anoikis, and pyroptosis. Here, we introduce a range of assays for measuring cell death in cultured cells, and we outline basic techniques for distinguishing healthy cells from apoptotic or necrotic cells-the two most common forms of cell death. We also provide personal insight into where these assays may be useful and how they may or may not be used to distinguish apoptotic cell death from other death modalities.

Measuring the DNA Content of Cells in Apoptosis and at Different Cell-Cycle Stages by Propidium Iodide Staining and Flow Cytometry.

All cells are created from preexisting cells. This involves complete duplication of the parent cell to create two daughter cells by a process known as the cell cycle. For this process to be successful, the DNA of the parent cell must be faithfully replicated so that each daughter cell receives a full copy of the genetic information. During the cell cycle, the DNA content of the parent cell increases as new DNA is synthesized (S phase). When there are two full copies of the DNA (G2/M phase), the cell splits to form two new cells (G0/G1 phase). As such, cells in different stages of the cell cycle have different DNA contents. The cell cycle is tightly regulated to safeguard the integrity of the cell and any cell that is defective or unable to complete the cell cycle is programmed to die by apoptosis. When this occurs, the DNA is fragmented into oligonucleosomal-sized fragments that are disposed of when the dead cell is removed by phagocytosis. Consequently apoptotic cells have reduced DNA content compared with living cells. This can be measured by staining cells with propidium iodide (PI), a fluorescent molecule that intercalates with DNA at a specific ratio. The level of PI fluorescence in a cell is, therefore, directly proportional to the DNA content of that cell. This protocol describes the use of PI staining to determine the percentage of cells in each phase of the cell cycle and the percentage of apoptotic cells in a sample.

Measuring Cell Death by Propidium Iodide Uptake and Flow Cytometry.

Propidium iodide (PI) is a small fluorescent molecule that binds to DNA but cannot passively traverse into cells that possess an intact plasma membrane. PI uptake versus exclusion can be used to discriminate dead cells, in which plasma membranes become permeable regardless of the mechanism of death, from live cells with intact membranes. PI is excited by wavelengths between 400 and 600 nm and emits light between 600 and 700 nm, and is therefore compatible with lasers and photodetectors commonly available in flow cytometers. This protocol for PI staining can be used to quantitate cell death in most modern research facilities and universities.

Cytometry in malaria--a practical replacement for microscopy?

Malaria, caused by protozoan Plasmodium parasites, kills ~800,000 people each year. Exact figures are uncertain because presumptive diagnoses are often made without identifying parasites in patients' blood either by microscopy, using Giemsa's century-old stain, or by simpler tests that are ultimately dependent on microscopy for quality control. Microscopy itself relies on trained observers' ability to detect subtle morphological features of parasitized red blood cells, only a few of which may be present on a slide. Quantitative and objective flow cytometric measurements of cellular constituents such as DNA, RNA, and the malaria pigment hemozoin are now useful in research in malaria biology and pharmacology, and can provide more reliable identification of parasite species and developmental stages and better detection of low-density parasitemia than could microscopy. The same measurements can now be implemented in much smaller, simpler, cheaper imaging cytometers, potentially providing a more accurate and precise diagnostic modality.

The Epstein-Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein.

The Epstein-Barr virus nuclear antigen (EBNA)-6 protein is essential for Epstein-Barr virus (EBV)-induced immortalization of primary human B-lymphocytes in vitro. In this study, fusion proteins of EBNA-6 with green fluorescent protein (GFP) have been used to characterize its nuclear localization and organization within the nucleus. EBNA-6 associates with nuclear structures and in immunofluorescence demonstrate a punctate staining pattern. Herein, we show that the association of EBNA-6 with these nuclear structures was maintained throughout the cell cycle and with the use of GFP-E6 deletion mutants, that the region amino acids 733-808 of EBNA-6 contains a domain that can influence the association of EBNA-6 with these nuclear structures. Co-immunofluorescence and confocal analyses demonstrated that EBNA-6 and EBNA-3 co-localize in the nucleus of cells. Expression of EBNA-6, but not EBNA-3, caused a redistribution of nuclear survival of motor neurons protein (SMN) to the EBNA-6 containing nuclear structures resulting in co-localization of SMN with EBNA-6.