Increased retention of functional mitochondria in mature sickle red blood cells is associated with increased sickling tendency, hemolysis and oxidative stress.

Abnormal retention of mitochondria in mature red blood cells (RBC) has been recently reported in sickle cell anemia (SCA) but their functionality and their role in the pathophysiology of SCA remain unknown. The presence of mitochondria within RBC was determined by flow cytometry in 61 SCA patients and ten healthy donors. Patients were classified according to the percentage of mature RBC with mitochondria contained in the whole RBC population: low (0-4%), moderate (>4% and <8%), or high level (>8%). RBC rheological, hematological, senescence and oxidative stress markers were compared between the three groups. RBC senescence and oxidative stress markers were also compared between mature RBC containing mitochondria and those without. The functionality of residual mitochondria in sickle RBC was measured by high-resolution respirometry assay and showed detectable mitochondrial oxygen consumption in sickle mature RBC but not in healthy RBC. Increased levels of mitochondrial reactive oxygen species were observed in mature sickle RBC when incubated with Antimycin A versus without. In addition, mature RBC retaining mitochondria exhibited greater levels of reactive oxygen species compared to RBC without mitochondria, as well as greater Ca2+, lower CD47 and greater phosphatidylserine exposure. Hematocrit and RBC deformability were lower, and the propensity of RBC to sickle under deoxygenation was higher, in the SCA group with a high percentage of mitochondria retention in mature RBC. This study showed the presence of functional mitochondria in mature sickle RBC, which could favor RBC sickling and accelerate RBC senescence, leading to increased cellular fragility and hemolysis.

L-asparaginase anti-tumor activity in pancreatic cancer is dependent on its glutaminase activity and resistance is mediated by glutamine synthetase.

l-Asparaginase is a cornerstone of acute lymphoblastic leukemia (ALL) therapy since lymphoblasts lack asparagine synthetase (ASNS) and rely on extracellular asparagine availability for survival. Resistance mechanisms are associated with increased ASNS expression in ALL. However, the association between ASNS and l-Asparaginase efficacy in solid tumors remains unclear, thus limiting clinical development. Interestingly, l-Asparaginase also has a glutaminase co-activity that is crucial in pancreatic cancer where KRAS mutations activate glutamine metabolism. By developing l-Asparaginase-resistant pancreatic cancer cells and using OMICS approaches, we identified glutamine synthetase (GS) as a marker of resistance to l-Asparaginase. GS is the only enzyme able to synthesize glutamine, and its expression also correlates with l-Asparaginase efficacy in 27 human cell lines from 11 cancer indications. Finally, we further demonstrated that GS inhibition prevents cancer cell adaptation to l-Asparaginase-induced glutamine starvation. These findings could pave the way to the development of promising drug combinations to overcome l-Asparaginase resistance.

Methionine tumor starvation by erythrocyte-encapsulated methionine gamma-lyase activity controlled with per os vitamin B6.

Erymet is a new therapy resulting from the encapsulation of a methionine gamma-lyase (MGL; EC number 4.4.1.11) in red blood cells (RBC). The aim of this study was to evaluate erymet potential efficacy in methionine (Met)-dependent cancers. We produced a highly purified MGL using a cGMP process, determined the pharmacokinetics/pharmacodynamics (PK/PD) properties of erymet in mice, and assessed its efficacy on tumor growth prevention. Cytotoxicity of purified MGL was tested in six cancer cell lines. CD1 mice were injected with single erymet product supplemented or not with vitamin B6 vitamer pyridoxine (PN; a precursor of PLP cofactor). NMRI nude mice were xenografted in the flank with U-87 MG-luc2 glioblastoma cells for tumor growth study following five intravenous (IV) injections of erymet with daily PN oral administration. Endpoints included efficacy and event-free survival (EFS). Finally, a repeated dose toxicity study of erymet combined with PN cofactor was conducted in CD1 mice. Recombinant MGL was cytotoxic on 4/6 cell lines tested. MGL half-life was increased from <24 h to 9-12 days when encapsulated in RBC. Conversion of PN into PLP by RBC was demonstrated. Combined erymet + PN treatment led to a sustained Met depletion in plasma for several days with a 85% reduction of tumor volume after 45 days following cells implantation, and a significant EFS prolongation for treated mice. Repeated injections in mice exhibited a very good tolerability with only minor impact on clinical state (piloerection, lean aspect) and a slight decrease in hemoglobin and triglyceride concentrations. This study demonstrated that encapsulation of methioninase inside erythrocyte greatly enhanced pharmacokinetics properties of the enzyme and is efficacy against tumor growth. The perspective on these results is the clinical evaluation of the erymet product in patients with Met starvation-sensitive tumors.

Innovative approach in Pompe disease therapy: Induction of immune tolerance by antigen-encapsulated red blood cells.

Pompe disease is a glycogen storage disease caused by acid α-glucosidase enzyme deficiency. Currently, the unique treatment is lifelong enzyme replacement therapy ERT with frequent intravenous administration of the recombinant analog alglucosidase-α (AGA), which ultimately generates a sustained humoral response resulting in treatment discontinuation. Our aim is to use the tolerogenic properties of antigen-encapsulated red blood cells (RBCs) to abolish the humoral response against AGA and to restore tolerance to replacement therapy. To demonstrate that our approach could prevent the AGA-induced immune response, mice were intravenously injected three times with AGA encapsulated into RBCs before being sensitized to AGA with several adjuvant molecules. Control animals received injections of free AGA instead of the encapsulated molecule. One-week after treatment with AGA-loaded RBCs, a strong decrease in specific humoral response was observed despite three stimulations with AGA and adjuvant molecules. Furthermore, this specific immunomodulation was maintained for at least two months without affecting the overall immune response. AGA-loaded RBCs represent a promising strategy to induce or restore tolerance in Pompe disease patients who develop hypersensitivity reactions following repeated AGA administrations.

Erythrocytes encapsulated with phenylalanine hydroxylase exhibit improved pharmacokinetics and lowered plasma phenylalanine levels in normal mice.

Enzyme replacement therapy is often hampered by the rapid clearance and degradation of the administered enzyme, limiting its efficacy and requiring frequent dosing. Encapsulation of therapeutic molecules into red blood cells (RBCs) is a clinically proven approach to improve the pharmacokinetics and efficacy of biologics and small molecule drugs. Here we evaluated the ability of RBCs encapsulated with phenylalanine hydroxylase (PAH) to metabolize phenylalanine (Phe) from the blood and confer sustained enzymatic activity in the circulation. Significant quantities of PAH were successfully encapsulated within murine RBCs (PAH-RBCs) with minimal loss of endogenous hemoglobin. While intravenously administered free PAH enzyme was rapidly eliminated from the blood within a few hours, PAH-RBCs persisted in the circulation for at least 10days. A single injection of PAH-RBCs was able to decrease Phe levels by nearly 80% in normal mice. These results demonstrate the ability of enzyme-loaded RBCs to metabolize circulating amino acids and highlight the potential to treat disorders of amino acid metabolism.

Red blood cells as innovative antigen carrier to induce specific immune tolerance.

The route of administration, the dose of antigen as well as the type of antigen-presenting cells (APCs) targeted are important factors to induce immune tolerance. Despite encouraging results obtained in animal models, intravenous injection of soluble antigen is unsuccessful in human clinical trials on autoimmune disease due to inefficient antigen delivery. To improve antigen delivery, we used mouse red blood cells (RBCs) as antigen vehicles to specifically target APCs which are responsible for removal of senescent RBCs after phagocytosis. In this study, we demonstrated that antigen-delivery by RBCs induced a strong decrease in the humoral response compared with the ovalbumin (OVA) free form in mice. In addition, OVA-loaded RBC treated with [bis(sulphosuccinimidyl)] suberate (BS3), a chemical compound known to enhance RBC phagocytosis, induced an inhibition of antigen-specific T cell responses and an increase in the percentage of regulatory T cells. The state of tolerance induced is long lasting, antigen-specific and sufficiently robust to withstand immunization with antigen mixed with cholera toxin adjuvant. This RBC strategy, which does not abolish the immune system, constitutes an attractive approach for induction of tolerance compared to systemic immunosuppressant therapies already in use.

Tumor growth control using red blood cells as the antigen delivery system and poly(I:C).

The goal of most current vaccines in tumor immunology is to induce an efficient immune response against the tumor cells. The use of red blood cells (RBCs) for the delivery of tumor-associated antigen to antigen-presenting cells is an innovative approach for cancer immunotherapy. The induction of antigen-specific immune responses after administration of antigen-loaded RBCs has been demonstrated previously in mice. In this paper, we show the utility of this delivery system for cancer immunotherapy in 2 tumor mouse models, using the E.G7-OVA and the B16F10 tumor cells. The non-self-antigen, ovalbumin, loaded in RBCs and the self-tumor antigen, tyrosinase-related protein 2, loaded in RBCs were tested in the E.G7-OVA and the B16F10 tumor models, respectively. We showed that not only protein but also peptide could be efficiently entrapped in RBCs by a controlled lysis/resealing process. In both antigen models, the administration of a small quantity of antigen loaded in RBCs combined with polyinosinic-polycytidylic acid induced an antigen-specific T-cell response and the control of tumor growth in mice, whereas the injection of the same quantity of free antigen did not. The intensity of the T-cell response was dependent on the concentrations of antigen entrapped and the treatment performed on the RBC membrane (antibody coating and heat treatment) to improve antigen delivery. In summary, these results support the use of RBCs as an antigen delivery system for a powerful cancer immunotherapy approach.

Pancreatic tumor sensitivity to plasma L-asparagine starvation.

OBJECTIVES: In this study, our aim was to test whether asparagine synthetase (ASNS) deficiency in pancreatic malignant cells can lead to sensitivity to asparagine starvation. We also investigated, in tumor-bearing mice, the efficacy of L-asparaginase entrapped in red blood cells (RBCs), a safe formulation, to induce asparagine depletion. METHODS: First, ASNS expression was evaluated by immunohistochemistry in sporadic pancreatic ductal adenocarcinoma. Then, 4 pancreatic carcinoma cell lines were examined by Western blot, immunocytochemistry, and cytotoxicity assay to L-asparaginase and in asparagine-free or reduced-asparagine media. Finally, mice bearing the most in vitro sensitive cell line received RBC-entrapped L-asparaginase to investigate the anticancer efficacy of serum asparagine depletion in vivo. RESULTS: Approximately 52% of pancreatic adenocarcinomas expressed no or low ASNS. The highest in vitro cytotoxicity to L-asparaginase or to reduced asparagine medium was observed with SW1990 line when ASNS expression was the lowest. In vivo sensitivity was confirmed for this cell line. CONCLUSIONS: Plasma asparagine depletion by RBC-entrapped L-asparaginase in selected patients having no low or no ASNS may be a promising therapeutic approach for pancreatic cancer.

Efficacy of homologous inositol hexaphosphate-loaded red blood cells in sickle transgenic mice.

Patients with sickle cell disease (SCD) can present several severe symptoms during their lifetime, including painful events due to vascular occlusion (VOC). Even though multiple factors are involved in VOC, hypoxia is the most important triggering factor. Inositol hexaphosphate (IHP) reduces the oxygen-haemoglobin affinity thus improving the oxygen release in the blood stream and in the tissues. Thus, IHP-loaded homologous red blood cells (IHP-RBCs) could be able to reduce disorders in SCD. The effectiveness of treatment was assessed in two types of SCD transgenic mice (BERK and SAD). The administration of four repeated injections of IHP-RBCs in BERK mice resulted in an improved survival rate and brain development, prevention of severe anaemia and a greatly lowered risk of VOC. After one injection of IHP-RBCs, SAD mice were subjected to acute hypoxic stress. Analysis of the lungs revealed significantly decreased mRNA levels of molecules involved in intravascular disorders. Our results showed that transfusion of homologous IHP-RBCs, by increasing the oxygen delivery, reduces SCD disorders in sickle transgenic mice.

A tuberculosis vaccine based on phosphoantigens and fusion proteins induces distinct gammadelta and alphabeta T cell responses in primates.

Phosphoantigens are mycobacterial non-peptide antigens that might enhance the immunogenicity of current subunit candidate vaccines for tuberculosis. However, their testing requires monkeys, the only animal models suitable for gammadelta T cell responses to mycobacteria. Thus here, the immunogenicity of 6-kDa early secretory antigenic target-mycolyl transferase complex antigen 85B (ESAT-6-Ag85B) (H-1 hybrid) fusion protein associated or not to a synthetic phosphoantigen was compared by a prime-boost regimen of two groups of eight cynomolgus. Although phosphoantigen activated immediately a strong release of systemic Th1 cytokines (IL-2, IL-6, IFN-gamma, TNF-alpha), it further anergized blood gammadelta T lymphocytes selectively. By contrast, the hybrid H-1 induced only memory alphabeta T cell responses, regardless of phosphoantigen. These latter essentially comprised cytotoxic T lymphocytes specific for Ag85B (on average + 430 cells/million PBMC) and few IFN-gamma-secreting cells (+ 40 cells/million PBMC, equally specific for ESAT-6 and for Ag85B). Hence, in macaques, a prime-boost with the H-1/phosphoantigen subunit combination induces two waves of immune responses, successively by gammadelta T and alphabeta T lymphocytes.

Developmental immunotoxicity investigations in the SD rat following pre- and post-natal exposure to cyclosporin.

BACKGROUND: The development and function of the immune system was assessed in juvenile SD rats following pre- or post-natal exposure to cyclosporin. The main objective was to assess the feasibility of the methods available for the detection of adverse effects on the development of the immune system for use in the safety assessment of medicines. METHODS: In a pre-natal experiment, 15 pregnant rats were given 10 mg/kg/day of cyclosporin by gavage from day 6 of gestation until 4 days after parturition. A control group received olive oil. In a post-natal experiment, the pups from 35 litters were given 10 mg/kg/day of cyclosporin by gavage from 4 to 28 days of age. Half of the pups in each litter were given water and acted as controls. Immune endpoints were determined in the pups in both experiments from two to 10 weeks of age, including: lymphocyte subsets, serum immunoglobulin titres, serum autoantibodies, primary antibody response to sheep red blood cells (SRBC), delayed-type hypersensitivity response, humoral response to keyhole limpet haemocyanin, spleen cellularity, immune organ weights, and histopathology. RESULTS: Pre-natal exposure caused no effects on immune function. Post-natal exposure caused immune depression during the treatment period and a persistent impairment of the immune system characterised by lymphoid hyperplasia in the spleen and a reduced primary antibody response to SRBC at 10 weeks of age. CONCLUSIONS: These results demonstrate the importance of a post-treatment follow-up period in developmental immunotoxicity studies, in order to distinguish between the transient effects of immune modulation and the persistent consequences of developmental toxicity.

Cytokine release does not improve the sensitivity and specificity of the direct popliteal lymph node assay.

The popliteal lymph node assay (PLNA) is being considered as a tool to predict the potential of drugs for inducing systemic autoimmune and hypersensitivity reactions. Despite the use of different technical approaches and the evaluation of over 130 compounds, the sensitivity and specificity of the PLNA are still debatable due to many false positive and negative responses. In this study, cytokine production was assessed as a possible endpoint to improve the direct (primary) PLNA. Diclofenac, imipramine, hydralazine, glafenin and minocycline were tested using the classical procedure. TH1 cytokines (IL-2 and IFN-gamma), TH2 cytokines (IL-4 and IL-5) and pro-inflammatory cytokines (IL-6, TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), IL-12p70 and IL-10) were measured in the serum and in suspensions of popliteal lymph node cells of female Balb/c mice by flow cytometry 7 days after drug administration. Only diclofenac and imipramine induced a cellularity index above 5 (considered as a positive response). Of the five tested drugs, only diclofenac induced a slight increase in TH1 cytokines, but there were no effects on TH2 cytokine production whatever the drug tested. Diclofenac increased the production of pro-inflammatory cytokines, whereas the production of MCP-1 was increased by minocycline and decreased by imipramine. No changes in serum cytokine levels were evident. These results suggest that measuring cytokine release is unlikely to improve the sensitivity and specificity of the direct PLNA.

Skin inflammation during contact hypersensitivity is mediated by early recruitment of CD8+ T cytotoxic 1 cells inducing keratinocyte apoptosis.

Contact hypersensitivity (CHS) is a T cell-mediated, Ag-specific skin inflammation induced by skin exposure to haptens in sensitized individuals. Th1/T cytotoxic 1 cells are effector cells of CHS, whereas Th2/T regulatory CD4(+) T cells have down-regulating properties. We have previously shown that CHS to 2,4-dinitrofluorobenzene is mediated by specific CD8(+) effector cells, whose cytolytic activity is mandatory for induction of skin inflammation. In this study, using immunohistochemistry and RT-PCR analysis, we show that CD8(+) T cells are rapidly recruited into the skin at the site of hapten challenge before the onset of clinical and histological signs of skin inflammation. This early CD8(+) T cell recruitment is concomitant with: 1) transient IFN-gamma mRNA expression suggesting local activation of effector cells; and 2) induction of keratinocyte (KC) apoptosis which gradually increased to a maximum at the peak of the CHS response. Alternatively, skin infiltration of CD4(+) T cells occurred later and coincided with the peak of the CHS reaction and the beginning of the resolution of skin inflammation. Mice deficient in CD8(+) T cells did not develop CHS, whereas mice deficient in CD4(+) T cells developed an enhanced inflammatory response with increased numbers of CD8(+) T cells recruited in the skin associated with massive KC apoptosis. These data show that CHS is due to the early and selective recruitment in the skin of CD8(+) T cytotoxic 1 effector cells responsible for KC apoptosis.