Isolation and Culture of Larval Cells from C. elegans

Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×104 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology

Effector and central memory T helper 2 cells respond differently to peptide immunotherapy

Peptide immunotherapy (PIT) offers realistic prospects for the treatment of allergic diseases, including allergic asthma. Much is understood of the behavior of naive T cells in response to PIT. However, treatment of patients with ongoing allergic disease requires detailed understanding of the responses of allergen-experienced T cells. CD62L expression by allergen-experienced T cells corresponds to effector/effector memory (CD62L(lo)) and central memory (CD62L(hi)) subsets, which vary with allergen exposure (e.g., during, or out with, pollen season). The efficacy of PIT on different T helper 2 (Th2) cell memory populations is unknown. We developed a murine model of PIT in allergic airway inflammation (AAI) driven by adoptively transferred, traceable ovalbumin-experienced Th2 cells. PIT effectively suppressed AAI driven by unfractionated Th2 cells. Selective transfer of CD62L(hi) and CD62L(lo) Th2 cells revealed that these two populations behaved differently from one another and from previously characterized (early deletional) responses of naive CD4(+) T cells to PIT. Most notably, allergen-reactive CD62L(lo) Th2 cells were long-lived within the lung after PIT, before allergen challenge, in contrast to CD62L(hi) Th2 cells. Despite this, PIT was most potent against CD62L(lo) Th2 cells in protecting from AAI, impairing their ability to produce Th2 cytokines, whereas this capacity was heightened in PIT-treated CD62L(hi) Th2 cells. We conclude that Th2 cells do not undergo an early deletional form of tolerance after PIT. Moreover, memory Th2 subsets respond differently to PIT. These findings have implications for the clinical translation of PIT in different allergic scenarios

Real-World Treatment Patterns, Clinical Outcomes, and Health Care Resource Utilization in Extensive-Stage Small Cell Lung Cancer in Canada

The prognosis for extensive-stage small cell lung cancer (ES-SCLC) is poor. Real-world evidence can highlight the unmet clinical need within this population. We conducted a population-based cohort study of ES-SCLC patients diagnosed in a large Canadian province (2010–2018) using electronic medical records and administrative claims data. In all, 1941 ES-SCLC patients were included, of which 476 (25%) were recurrent cases. Median age at diagnosis was 70 years (range: 39–94) and 50.2% were men. Of the 1941 ES-SCLC patients, 29.5% received chemotherapy and radiotherapy, 17.0% chemotherapy alone, 8.7% radiotherapy alone, and 44.8% received best supportive care. Chemotherapy was initiated by 46.5%, 8.5%, and 1.4% of first-, second-, and third-line patients, with lower uptake for recurrent cases. Median survival from first-, second-, and third-line chemotherapy was 7.82 months (95% CI: 7.50–8.22), 5.72 months (95% CI: 4.90–6.87), and 3.83 months (95% CI: 2.99–4.60). Among patients who received first-line therapy, the 2-year and 5-year survival was 7.3% (95% CI: 5.7–9.2) and 2.9% (95% CI: 1.8–4.5). In conclusion, initiation of first-line treatment in ES-SCLC was low with significant attrition in subsequent lines. These results underscore the need for effective front-line treatments and highlight the potential for novel therapies to improve patient outcomes