Disposition of treosulfan and its active monoepoxide in a bone marrow, liver, lungs, brain, and muscle: Studies in a rat model with clinical relevance.

For the recent years, the application of treosulfan (TREO)-based conditioning prior to hematopoietic stem cell transplantation (HSCT) has been increasing as an alternative to busulfan-based therapy, especially for patients presenting high risk of developing hepato-, pulmo-, and neurotoxicity. So far, the penetration of TREO and its epoxy-derivatives into central nervous system and aqueous humor of the eye has been investigated. However, lacking knowledge on the compounds distribution into the other key tissues precludes comprehensive understanding and assessment of TREO clinical efficacy and toxicity. In this paper, the disposition of TREO and its active monoepoxide (S,S-EBDM) in a bone marrow, liver, lungs, brain, and quadriceps femoris was studied in an animal model. Male and female adult Wistar rats (n=48/48) received an intraperitoneal injection of TREO at the dose of 500mg/kg b.w. Concentrations of TREO and S,S-EBDM in tissues were determined with a validated HPLC-MS/MS method. Pharmacokinetic calculations were performed in WinNonlin using a noncompartmental analysis. Mean values of the maximal concentrations of TREO and S,S-EBDM in the organs were sex-independent and ranged from 61 to 1650µM and 25-105µM, respectively. No quantifiable levels of S,S-EBDM were found in the liver. Average tissue/plasma area under the curve (AUC) ratio for unbound TREO increased in the sequence: brain (0.10)

Ascorbic acid serum levels are reduced in patients with hematological malignancies.

In this paper we demonstrate that patients treated with chemotherapy and/or hematopoietic stem cell transplantation (HSCT) have highly significant reduced serum ascorbic acid (AA) levels compared to healthy controls. We recently observed in in vitro experiments that growth of both T and NK cells from hematopoietic stem cells is positively influenced by AA. It might be of clinical relevance to study the function and recovery of immune cells after intensive treatment, its correlation to AA serum levels and the possible effect of AA supplementation.

Tattoo Delivery of a Semliki Forest Virus-Based Vaccine Encoding Human Papillomavirus E6 and E7.

The skin is an attractive organ for immunization because of the presence of antigen-presenting cells. Intradermal delivery via tattooing has demonstrated superior vaccine immunogenicity of DNA vaccines in comparison to conventional delivery methods. In this study, we explored the efficacy of tattoo injection of a tumor vaccine based on recombinant Semliki Forest virus replicon particles (rSFV) targeting human papillomavirus (HPV). Tattoo injection of rSFV particles resulted in antigen expression in both the skin and draining lymph nodes. In comparison with intramuscular injection, the overall antigen expression determined at the site of administration and draining lymph nodes was 10-fold lower upon tattoo injection. Delivery of SFV particles encoding the E6 and E7 antigens of human papillomavirus type 16 (SFVeE6,7) via tattooing resulted in HPV-specific cytotoxic T cells and in vivo therapeutic antitumor response. Strikingly, despite the observed lower overall transgene expression, SFVeE6,7 delivered via tattoo injection resulted in higher or equal levels of immune responses as compared to intramuscular injection. The intrinsic immunogenic potential of tattooing provides a benefit for immunotherapy based on an alphavirus.

Ascorbic acid promotes proliferation of natural killer cell populations in culture systems applicable for natural killer cell therapy.

BACKGROUND AIMS: Natural killer (NK) cell-based immunotherapy is a promising treatment for a variety of malignancies. However, generating sufficient cell numbers for therapy remains a challenge. To achieve this, optimization of protocols is required. METHODS: Mature NK cells were expanded from peripheral blood mononuclear cells PBMCs in the presence of anti-CD3 monoclonal antibody and interleukin-2. Additionally, NK-cell progenitors were generated from CD34(+) hematopoietic stem cells or different T/NK-cell progenitor populations. Generated NK cells were extensively phenotyped, and functionality was determined by means of cytotoxicity assay. RESULTS: Addition of ascorbic acid (AA) resulted in more proliferation of NK cells without influencing NK-cell functionality. In more detail, PBMC-derived NK cells expanded 2362-fold (median, range: 90-31,351) in the presence of AA and were capable of killing tumor cells under normoxia and hypoxia. Moreover, hematopoietic stem cell-derived progenitors appeared to mature faster in the presence of AA, which was also observed in the NK-cell differentiation from early T/NK-cell progenitors. CONCLUSIONS: Mature NK cells proliferate faster in the presence of phospho-L-AA, resulting in higher cell numbers with accurate functional capacity, which is required for adoptive immunotherapy.

Sequential intranodal immunotherapy induces antitumor immunity and correlated regression of disseminated follicular lymphoma.

Advanced stage follicular lymphoma (FL) is incurable by conventional therapies. In the present pilot clinical trial, we explored the efficacy and immunogenicity of a novel in situ immunotherapeutic strategy. Fourteen patients with untreated or relapsed stage III/IV FL were included and received local radiotherapy to solitary lymphoma nodes and intranodal injections of low-dose rituximab (5 mg), immature autologous dendritic cells, and granulocyte-macrophage colony-stimulating factor at the same site. The treatment was repeated 3 times targeting different lymphoma nodes. Primary end points were clinical responses and induction of systemic immunity. Five out of 14 patients (36%) displayed objective clinical responses, including 1 patient with cutaneous FL who showed regression of skin lesions. Two of the patients had durable complete remissions. Notably, the magnitude of vaccination-induced systemic CD8 T-cell-mediated responses correlated closely with reduction in total tumor area (r = 0.71, P = .006), and immune responders showed prolonged time to next treatment. Clinical responders did not have a lower tumor burden than nonresponders pretreatment, suggesting that the T cells could eliminate large tumor masses once immune responses were induced. In conclusion, the combined use of 3 treatment modalities, and in situ administration in single lymphoma nodes, mediated systemic T-cell immunity accompanied by regression of disseminated FL. The trial was registered at www.clinicaltrials.gov as #NCT01926639.

Technical advance: ascorbic acid induces development of double-positive T cells from human hematopoietic stem cells in the absence of stromal cells.

The efficacy of donor HSCT is partly reduced as a result of slow post-transplantation immune recovery. In particular, T cell regeneration is generally delayed, resulting in high infection-related mortality in the first years post-transplantation. Adoptive transfer of in vitro-generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled, feeder-free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect, indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore, the addition of AA to feeder cocultures resulted in development of DP and SP T cells, whereas without AA, a preT cell-stage arrest occurred. We conclude that neither DLL4-expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G-CSF-mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy.

Therapeutic immunization and local low-dose tumor irradiation, a reinforcing combination.

Therapeutic cancer vaccines show promise in preclinical studies, yet their clinical efficacy is limited. Increased recruitment of immune cells into tumors and suppression of the immune suppressive tumor environment are critical components toward effective cancer immunotherapies. Here, we report how local low-dose irradiation, alone or with a therapeutic immunization based on Semliki Forest virus (SFV) against human papillomavirus (HPV)-related cancer, influences these immune mechanisms. We first demonstrated that immunization with SFVeE6,7 or SFVeOVA, replicon particles expressing either HPV16 E6/E7 or ovalbumin, resulted in an antigen-specific migration of CD8+ T cells into HPV- and OVA-specific tumors. Local low-dose tumor irradiation alone resulted in a 2-fold increase of intratumoral CD8+ T cells. When 14 Gy irradiation was combined with immunization, intratumoral numbers of CD8+ T cells increased 10-fold and the number of CD8+ T cells specific for the E7- epitope increased more than 20-fold. Irradiation alone however also increased the number of intratumoral myeloid-derived suppressor cells (MDSCs) 3.5-fold. Importantly, this number did not further increase when combined with immunization. As a result, the ratio of antigen-specific CD8+ T cells and MDSCs in tumors increased up to 85-fold compared to the control. We furthermore demonstrated that following irradiation CCR2 and CCL2, CXCR6 and CCL16, chemokines and ligands involved in tumor homing of immune cells, were significantly up regulated. This study demonstrates that local low-dose tumor irradiation influences the intratumoral immune population induced by SFVeE6,7 immunization by a strong increase in the ratio of antitumoral to immune suppressive cells, thus changing the intratumoral immune balance in favor of antitumor activity.

Role of regulatory T-cells in immunization strategies involving a recombinant alphavirus vector system.

BACKGROUND: Regulatory T-cells (Treg) hamper immune responses elicited by cancer vaccines. Therefore, depletion of Treg is being used to improve the outcome of vaccinations. METHODS: We studied whether an alphavirus vector-based immunotherapeutic vaccine changes the number and/or activity of Treg and if Treg depletion improves the efficacy of this vaccine against tumours. The vaccine is based on a Semliki Forest virus (SFV). The recombinant SFV replicon particles encode a fusion protein of E6 and E7 from human papillomavirus (HPV) type 16 (SFVeE6,7). RESULTS: We demonstrated that SFVeE6,7 immunization did not change Treg levels and their suppressive activity. Depletion of Treg in mice, using the novel anti-folate receptor 4 antibody, did not enhance the immune response induced by SFVeE6,7 immunization. Both the priming and the proliferation phases of the HPV-specific response elicited with SFVeE6,7 were not affected by the immune-suppressive activity of Treg. Moreover, Treg depletion did not improve the therapeutic antitumour response of SFVeE6,7 in a murine tumour model. CONCLUSIONS: The efficacy of the SFVeE6,7 vaccine was not hampered by Treg. Therefore, SFVeE6,7 seems a very promising candidate for the treatment of HPV-induced disease, as it may not require additional immune interventions to modulate Treg activity.

Heterologous prime-boost immunizations with a virosomal and an alphavirus replicon vaccine.

Heterologous prime-boost immunization strategies in general establish higher frequencies of antigen-specific T lymphocytes than homologous prime-boost protocols or single immunizations. We developed virosomes and recombinant Semliki Forest virus (rSFV) as antigen delivery systems, each capable of inducing strong CTL responses in homologous prime-boost protocols. Here, we demonstrate that a heterologous prime-boost with recombinant Semliki Forest virus (rSFV) encoding a fusion protein of E6 and E7 of human papillomavirus (HPV) type 16 and virosomes containing the HPV16 E7 protein resulted in higher numbers of antigen-specific CTL in mice than homologous protocols. Evasion of vector-specific immunity appeared to play a role in establishing these high frequencies, as coinduction of vector-specific responses during the prime immunization reduced the frequency of antigen-specific CTL after a heterologous booster. However, the high numbers of CTL initially primed by the heterologous protocols did not correlate with enhanced responsiveness to in vitro antigenic stimulation, nor in improved cytolytic activity or antitumor responses in vivo compared to a homologous protocol with rSFV. This lack of correlation could not be explained by changes in numbers of regulatory T cells. However, we observed differences in the frequencies of T cell subsets within the E7-specific CD8(+) T cell population, e.g. higher frequencies of central memory T cells upon homologous immunizations compared to heterologous immunizations. The induction of central memory T cells is crucial for a cancer vaccine as these cells are known to rapidly expand upon recall stimulation. This study demonstrates that the strongly increased number of antigen-specific CTL as induced by heterologous prime-boost immunizations, often used as a proof for the enhanced efficacy of such regimes, does not necessarily equal superior functional antitumor responses.

[Pharmacological mechanisms of Doxorubicin activity (DOX) - current state of knowledge]. / Mechanizmy dzialania doksorubicyny (DOX)--obecny stan wiedzy.

Doxorubicin (DOX) belongs to the anthracycline antibiotics, widely used in the treatment of various types of malignancies. This review presents recent data about mechanisms of the drug action.