Absolute lymphocyte count proliferation kinetics after CAR T-cell infusion impact response and relapse

CD19-directed chimeric antigen receptor (CAR) T cells show characteristic proliferation kinetics after infusion that correlate with response. Clearance of circulating disease, B-cell aplasia (BCA), and cytokine release syndrome (CRS) are used to observe CAR T-cell function, given the lack of commercial CAR T-cell measurement assays. We investigated the utility of common hematology laboratory parameters in 166 patients with B-cell acute lymphoblastic leukemia (B-ALL) who were treated with CAR T-cell therapy targeting CD19. CAR T-cell infusion was followed by disappearance of circulating blasts in 86% of patients at a median of 6 days. After a lag phase, there was a rapid expansion in absolute lymphocyte count (ALC) in the second week that coincided with the appearance of atypical lymphocytes. The expansion phase was followed by a contraction phase with a concomitant decrease in atypical lymphocytes. In vitro CAR T-cell studies showed similar kinetics and morphological changes. Peak ALC and overall expansion was greater in sustained responders compared with that in nonresponders. Patients with early loss of BCA and those with eventual CD19+ minimal residual disease/relapse showed lower overall lymphocyte expansion compared with the controls. Pleomorphic lymphocytosis was noted in the cerebrospinal fluid at post-CAR time points. We conclude that lymphocyte counts and differential can also be used to evaluate CAR T-cell expansion after infusion, along with BCA and CRS. This is the first report to characterize the morphology of CAR T cells and determine the utility of lymphocyte kinetics.

Lytic cycle of Besnoitia besnoiti tachyzoites displays similar features in primary bovine endothelial cells and fibroblasts

Background Bovine besnoitiosis, caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that continues to spread in Europe in the absence of control tools. In this scenario, in vitro culture systems are valuable tools to carry out drug screenings and to unravel host-parasite interactions. However, studies performed in bovine target cells are scarce. Methods The objective of the present study was to obtain primary bovine aortic endothelial cells (BAECs) and fibroblast cell cultures, target cells during the acute and the chronic stage of the disease, respectively, from healthy bovine donors. Afterwards, expression of surface (CD31, CD34 and CD44) and intracellular markers (vimentin and cytokeratin) was studied to characterize cell populations by flow cytometry. Next, the lytic cycle of B. besnoiti tachyzoites was studied in both target cells. Invasion rates (IRs) were determined by immunofluorescence at several time points post-infection, and proliferation kinetics were studied by quantitative PCR (qPCR). Finally, the influence of bovine viral diarrhea virus (BVDV) co-infection on the host cell machinery, and consequently on B. besnoiti invasion and proliferation, was investigated in BAECs. Results Morphology and cytometry results confirmed the endothelial and fibroblast origins. CD31 was the surface marker that best discriminated between BAECs and fibroblasts, since fibroblasts lacked CD31 labelling. Expression of CD34 was weak in low-passage BAECs and absent in high-passage BAECs and fibroblasts. Positive labelling for CD44, vimentin and cytokeratin was observed in both BAECs and fibroblasts. Regarding the lytic cycle of the parasite, although low invasion rates (approximately 3–4%) were found in both cell culture systems, more invasion was observed in BAECs at 24 and 72 hpi. The proliferation kinetics did not differ between BAECs and fibroblasts. BVDV infection favoured early Besnoitia invasion but there was no difference in tachyzoite yields observed in BVDV-BAECs compared to BAECs. Conclusions We have generated and characterized two novel standardized in vitro models for Besnoitia besnoiti infection based on bovine primary target BAECs and fibroblasts, and have shown the relevance of BVDV coinfections, which should be considered in further studies with other cattle pathogens.

Effectiveness of Pleurotus ostreatus Extract Through Cytotoxic Test and Apoptosis Mechanism of Cervical Cancer Cells

<p><em>Pleurotus ostreatus</em> is a common mushroom cultivated in Indonesia, and potential properties of bioactive compounds for medicinal mushroom. This study was aimed at obtaining <em>P.ostreatus</em> extract bioactive compounds potential in inhibiting the proliferation of cervical cancer cells (HeLa) and evaluating the HeLa cell proliferation kinetics and HeLa cell death mechanisms. The research was beneficial in making this product can be easily applied in a more controlled industrial scale. Anticancer activity test through a cytotoxic test using the MTT [3- (4,5-dimetiltiazol-2-yl) -2.5-diphenyl tertrazolium bromide], the kinetics proliferation of HeLa cells and HeLa cell death mechanism was performed. Linear regression analysis was used to analyze the data. Ethyl acetate extract of <em>P.</em> <em>ostreatus</em> isolated from Madiun showed the best results with IC ₅₀ = 107.59 µg / ml. HeLa cell proliferation kinetics analysis showed that the application of bioactive compounds 100 µg / ml resulted in an increase of in death of HeLa cells along with length of incubation time. An important finding was that HeLa cells death by apoptosis was greater than by necrosis. In conclusion, the extracts of <em>P.</em> <em>ostreatus</em> has the potential to inhibit the growth of HeLa cells.</p><p> </p>