Lytic efficiency of immunosuppressive drug-resistant armoured T cells against circulating HBV-related HCC in whole blood.

Recurrence of hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) after liver transplant (LT) is mediated by circulating tumour cells (CTCs) and exacerbated by the immunosuppressants required to prevent graft rejection. To circumvent the effects of immunosuppressants, we developed immunosuppressive drug-resistant armoured HBV-specific T-cell receptor-redirected T cells (IDRA HBV-TCR). However, their ability to eliminate HBV-HCC circulating in the whole blood has never been tested, and whether their lytic efficacy is compatible with the number of adoptively transferred T cells in vivo has never been measured. Hence, we developed a microscopy-based assay to quantify CTCs in whole blood. The assay was then used to quantify the efficacy of IDRA HBV-TCRs to lyse free-floating HBV-HCC cells in the presence of Tacrolimus and Mycophenolate Mofetil (MMF). We demonstrated that a panel of antibodies (AFP, GPC3, Vimentin, pan-Cytokeratin, and CD45) specific for HCC tumour antigens and immune cells can effectively differentiate HCC-CTCs in whole blood. Through dose-titration experiments, we observed that in the presence of immunosuppressive drugs, a minimum of 20 000 IDRA HBV-TCR T cells/ml of whole blood is necessary to lyse ~63.5% of free-floating HBV-HCC cells within 16 hours. In conclusion, IDRA HBV-TCR T cells can lyse free-floating HBV-HCC cells in whole blood in the presence of Tacrolimus and MMF. The quantity of IDRA-HBV TCR T cells required can be achieved by the adoptive transfer of 5 × 106 IDRA-HBV TCR-T cells/kg, supporting the utilisation of IDRA HBV-TCR T cells to eliminate CTCs as prophylaxis against recurrence after LT.

Cell-Main Spectra Profile Screening Technique in Simulation of Circulating Tumour Cells Using MALDI-TOF Mass Spectrometry.

Circulating atypical cells (CAC) are released from a primary tumour site into peripheral blood and are indicators of cancer metastasis. CAC occur at very low frequency in circulating blood, and their detection remains challenging. Moreover, white blood cells (WBC) are the major contaminant in enriched CAC samples. Here, we developed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a novel CAC characterization platform. Main spectra profiles (MSP) of normal and cancer cells were generated by MALDI-TOF MS, and a cell-main spectra database was then compiled and analysed using the MALDI Biotyper software. Logarithmic scores accurately predicted distinct cell types. The feasibility of this workflow was then validated using simulated samples, which were prepared by 5000 WBC of three healthy individuals spiked with varying numbers (3, 6, 12, 25, 50, and 100) of lung, colon, or prostate cancer cells. MALDI-TOF MS was able to detect cancer cells down to six cells over the background noise of 5000 WBC with significantly higher predictive scores as compared to WBC alone. Further development of cell-MSP database to cover all cancer types sourced from cell lines and patient tumours may enable the use of MALDI-TOF MS as a cancer-screening platform in clinical settings in the future.

Accuracy of Tumour-Associated Circulating Endothelial Cells as a Screening Biomarker for Clinically Significant Prostate Cancer.

Even though more than 350,000 men die from prostate cancer every year, broad-based screening for the disease remains a controversial topic. Guidelines demand that the only commonly accepted screening tool, prostate-specific antigen (PSA) testing, must be followed by prostate biopsy if results are elevated. Due to the procedure's low positive predictive value (PPV), however, over 80% of biopsies are performed on healthy men or men with clinically insignificant cancer-prompting calls for new ways of vetting equivocal PSA readings prior to the procedure. Responding to the challenge, the present study investigated the diagnostic potential of tumour-associated circulating endothelial cells (tCECs), which have previously been described as a novel, blood-based biomarker for clinically significant cancers. Specifically, the objective was to determine the diagnostic accuracy of a tCEC-based blood test to detect clinically significant prostate cancer (defined as Gleason score ≥ 3 + 4) in high-risk patients. Performed in a blinded, prospective, single-centre set-up, it compared a novel tCEC index test with transrectal ultrasound-guided biopsy biopsy as a reference on a total of 170 patients and found that a tCEC add-on test will almost double the PPV of a standalone PSA test (32% vs. 17%; p = 0.0012), while retaining a negative predictive value above 90%.

Malarial anemia: digestive vacuole of Plasmodium falciparum mediates complement deposition on bystander cells to provoke hemophagocytosis.

The digestive vacuole (DV) of Plasmodium falciparum, which is released into the bloodstream upon rupture of each parasitized red blood cell (RBC), was recently discovered to activate the alternative complement pathway. In the present work, we show that C3- and C5-convertases assembling on the parasitic organelle are able to provoke deposition of activated C3 and C5b-9 on non-infected bystander erythrocytes. Direct contact of DVs with cells is mandatory for the effect, and bystander complement deposition occurs focally, possibly at the sites of contact. Complement opsonization promotes protracted erythrophagocytosis by human macrophages, an effect that is magnified when ring-stage infected RBCs with reduced CD55 and CD59, or paroxysmal nocturnal hemoglobinuria (PNH)-RBCs lacking these complement inhibitors are employed as targets. Bystander attack can also directly induce lysis of PNH-RBCs. Direct evidence for complement activation and bystander attack mediated by DVs was obtained through immunohistochemical analyses of brain paraffin sections from autopsies of patients who had died of cerebral malaria. C3d and the assembled C5b-9 complex could be detected in all sections, colocalizing with and often extending locally beyond massive accumulations of DVs that were identified under polarized light. This is the first demonstration that a complement-activating particle can mediate opsonization of bystander cells to promote their antibody-independent phagocytosis. The phenomenon may act in concert with other pathomechanisms to promote the development of anemia in patients with severe malaria.

Digestive vacuole of Plasmodium falciparum released during erythrocyte rupture dually activates complement and coagulation.

Severe Plasmodium falciparum malaria evolves through the interplay among capillary sequestration of parasitized erythrocytes, deregulated inflammatory responses, and hemostasis dysfunction. After rupture, each parasitized erythrocyte releases not only infective merozoites, but also the digestive vacuole (DV), a membrane-bounded organelle containing the malaria pigment hemozoin. In the present study, we report that the intact organelle, but not isolated hemozoin, dually activates the alternative complement and the intrinsic clotting pathway. Procoagulant activity is destroyed by phospholipase C treatment, indicating a critical role of phospholipid head groups exposed at the DV surface. Intravenous injection of DVs caused alternative pathway complement consumption and provoked apathy and reduced nociceptive responses in rats. Ultrasonication destroyed complement-activating and procoagulant properties in vitro and rendered the DVs biologically inactive in vivo. Low-molecular-weight dextran sulfate blocked activation of both complement and coagulation and protected animals from the harmful effects of DV infusion. We surmise that in chronic malaria, complement activation by and opsonization of the DV may serve a useful function in directing hemozoin to phagocytic cells for safe disposal. However, when the waste disposal system of the host is overburdened, DVs may transform into a trigger of pathology and therefore represent a potential therapeutic target in severe malaria.

Digestive vacuoles of Plasmodium falciparum are selectively phagocytosed by and impair killing function of polymorphonuclear leukocytes.

Sequestration of parasitized erythrocytes and dysregulation of the coagulation and complement system are hallmarks of severe Plasmodium falciparum malaria. A link between these events emerged through the discovery that the parasite digestive vacuole (DV), which is released together with infective merozoites into the bloodstream, dually activates the intrinsic clotting and alternative complement pathway. Complement attack occurs exclusively on the membrane of the DVs, and the question followed whether DVs might be marked for uptake by polymorphonuclear granulocytes (PMNs). We report that DVs are indeed rapidly phagocytosed by PMNs after schizont rupture in active human serum. Uptake of malaria pigment requires an intact DV membrane and does not occur when the pigment is extracted from the organelle. Merozoites are not opsonized and escape phagocytosis in nonimmune serum. Antimalarial Abs mediate some uptake of the parasites, but to an extent that is not sufficient to markedly reduce reinvasion rates. Phagocytosis of DVs induces a vigorous respiratory burst that drives the cells into a state of functional exhaustion, blunting the production of reactive oxygen species (ROS) and microbicidal activity upon challenge with bacterial pathogens. Systemic overloading of PMNs with DVs may contribute to the enhanced susceptibility of patients with severe malaria toward invasive bacterial infections.

Re-evaluating acridine orange for rapid flow cytometric enumeration of parasitemia in malaria-infected rodents.

Methods facilitating research in malaria are of pivotal relevance. Flow cytometry offers the possibility of rapid enumeration of parasitemia. It relies on staining the parasite DNA to distinguish between infected and non-infected red blood cell (RBC) populations. Unfortunately, in rodents abundant reticulocyte RNA interferes with the application of the method. This results in time-consuming sample preparation protocols that offer no clear advantage over microscopic counting. We re-evaluated the use of the DNA/RNA discriminating vital fluorochrome acridine orange (AO) for rapid flow cytometric enumeration of parasitemia in rodents. Whole blood from rodents infected with Plasmodium berghei and Plasmodium yoelii was stained with AO and analyzed by flow cytometer. A newly developed two-channel (FL1/FL3) detection method was compared with conventional one-channel (FL1) detection and microscopic counting. The new AO two-channel detection method clearly discriminated between infected and non-infected RBC populations. It showed to be linear above parasitemias of 0.3%. Sample processing time amounted to approximately 5 min. It is shown that AO can be used for rapid, precise, and accurate enumeration of parasitemia in rodents. Due to its ease of handling the method might find widespread application in malaria research.