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We assessed the benefit of predeposite autologous blood donation (PAD) before bone marrow (BM) donation on transfusion requirements, haemoglobin concentrations (Hb) and the occurrence of adverse events (AE). We collected data retrospectively from 50 donors of BM with PAD from 2010 to 2014. An autologous transfusion (AT) was given to 50% of the donors (group 1). In the group 2, the products from PAD were not used. The total volume median of marrow harvested was 17.7 mL/k (range 12.3-21.4) in the group 1 and 13.3 mL/k (8.6-22.6) in the group 2. The female ratio was higher in the group 1 (60%) than in the group 2 (16%). Bone marrow harvest led to a decline in Hb (from PAD to first day after BM donation) by 2.9 g/dL (1.5-5.5) in the group 1 and by 3.5 g/dL (1.2-5) in the group 2. The post-harvest Hb (D+1) median was identical in the two groups: 10.9 g/dL (7.6-13.5) in the group 1 versus 11.5 g/dL (9.3-13.4) in the group 2. Six AE were reported in each group. In the group with AE, the median weight was lower: 58 k (50-71) versus 75 k (52-110); and the median total volume of marrow harvested was higher: 20.1 mL/k (9.9-21.4) versus 14.3 mL/k (8.6-22.6). All post-harvest Hb were ≥ 7.6g/dL. This study shows the high loss of Hb after BM donation but not enough to prove a blood transfusion in BM donors with median age of 36 years (16-62) and without comorbidity. The occurrence of AE (25% of BM donors) justifies a careful surveillance after the BM donation. The PAD should not be routinely offered to bone marrow donors., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

This study compares the behavior of 2 commercially available polyriboinosinic-polyribocytidylic acids (poly I:C1 and poly I:C2) and the structural analog poly I:C12U in regard to dendritic cell (DC) maturation. When the Toll-like receptor 3 (TLR3) agonists are tested in combination with interferon-alpha, tumor necrosis factor-alpha, interleukin (IL)-1beta, and interferon-gamma (the so-called alpha-type-1 DC), the 3 different cocktails generate phenotypically mature DCs, but with different functional properties. Higher migratory capacity is observed with poly I:C1, the only poly I:C allowing spontaneous release of IL-12p70 by DCs. However, upon CD40 triggering, cocktails containing poly I:C2 or poly I:C12U allow a far higher production of IL-12p70 compared with those containing poly I:C1. Using a TLR signaling pathway reverse transcription profiler polymerase chain reaction to analyze changes in gene expression after treatment of DCs with the agonists alone, we show that 39% of the 84 tested genes are differentially regulated between the 3 conditions. Poly I:C12U induces far fewer regulated genes than the 2 other poly I:Cs. These different behaviors could be due to alternative ways of sensing double-stranded RNA, which do not rely solely on TLR3 but also on other types of receptors, depending on the size of poly I:Cs. As the 2 poly I:Cs tested here have very different molecular weights, this could partly explain the observed differences. In conclusion, neither the poly I:Cs nor their structural analog poly I:C12U have an equivalent behavior. This should be taken into an account not only when they are used in cocktails for DC maturation but also when analyzing signaling pathways with synthetic double-stranded RNA analogs.

Prokineticin 1 and 2 (PROK1 and PROK2) are two small proteins largely expressed in inflammatory tissues and involved in monocyte activation and differentiation. The focus of this study was to evaluate whether PROK1 was able to induce chemokine secretion in human monocytes, in monocyte-derived macrophages and in monocyte-derived dendritic cells, an aspect not addressed thus far. Here, we show for the first time, using flow cytometry, that PROK receptors 1 and 2 are present on the surface of human monocytes. Subsequently, monocytes were selected to investigate the chemokine response after stimulation by PROK1. Our results show that only three chemokines (CCL4, CXCL1 and CXCL8) were significantly induced at both the transcript and protein level, and that PROK1 induces most potently CXCL8, in a dose-dependent manner. From a mechanistic point of view, by blocking independently Galphai protein or intracellular calcium, monocytes lose the ability to secrete CXCL8 in response to PROK1. Finally, we observed that CCL4, CXCL1 and CXCL8 secretion, following PROK1 induction, is only observed in monocytes and not in monocyte-derived macrophages and dendritic cells. Our results demonstrate that, in vitro, the differentiation status of monocytes influences chemokine production after stimulation by PROK1, and that this chemokine production is geared toward a pro-inflammatory response. This could represent a novel amplification loop of leukocyte recruitment, extravasation and tissue invasion.

Purpose: The purpose of this study was to investigate the biodistribution of mature dendritic cells (DCs) injected by various routes, during a cell therapy protocol., Methods: In the context of a vaccine therapy protocol for melanoma, DCs matured with Ribomunyl and interferon-gamma were labelled with( 111)In-oxine and injected into eight patients along various routes: afferent lymphatic vessel (IL) (4 times), lymph node (IN) (5 times) and intradermally (ID) (6 times)., Results: Scintigraphic investigations showed that the IL route allowed localisation of 80% of injected radioactivity in eight to ten nodes. In three cases of IN injection, the entire radioactivity stagnated in the injected nodes, while in two cases, migration to adjacent nodes was observed. This migration was detected rapidly after injection, as with IL injections, suggesting that passive transport occurred along the physiological lymphatic pathways. In two of the six ID injections, 1-2% of injected radioactivity reached a proximal lymph node. Migration was detectable in the first hour, but increased considerably after 24 h, suggesting an active migration mechanism. In both of the aforementioned cases, DCs were strongly CCR7-positive, but this feature was not a sufficient condition for effective migration. In comparison with DCs matured with TNF-alpha, IL-1beta, IL-6 and PGE2, our DCs showed a weaker in vitro migratory response to CCL21, despite comparable CCR7 expression, and higher allostimulatory and TH1 polarisation capacities., Conclusion: The IL route allowed reproducible administration of specified numbers of DCs. The IN route sometimes yielded fairly similar results, but not reproducibly. Lastly, we showed that DCs matured without PGE2 that have in vitro TH1 polarisation capacities can migrate to lymph nodes after ID injection.

Patients' autologous macrophages (AM) were used as antigen-presenting cells (APC) in a vaccination protocol against malignant melanoma. AM were administered by various routes, including intralymphatic, since these cells did not express CCR7, a molecule required for APC migration to lymph nodes. Seven HLA-A2 patients with metastatic melanoma-two classified as M1 and five as M3-were included in the study. AM were produced from leukapheresis-separated mononuclear cells by 7-day culture with granulocyte-macrophage colony-stimulating factor. After separation by elutriation, AM were frozen in aliquots and subsequently thawed at monthly intervals, exposed to MAGE-3(271-279) peptide and injected subcutaneously into lymph nodes or into one peripheral lymph vessel. Intradermal tests were performed before and after treatment to determine peptide reactivity. No acute toxicity was observed following injection. One M1 patient had a 7-mm induration intradermal reaction response and was stabilized for 64 weeks. The M3 patients did not show any immunological or clinical response. In 11 patients, the biodistribution of 111In-labeled AM was investigated. There was no clear evidence that AM injected intradermally or subcutaneously left the site of injection. After injection into a lymph vessel of the foot region, scintigraphs showed five to ten popliteal and inguinocrural lymph nodes. This appeared to be the most efficient way to administer rapidly and safely large amounts of peptide-loaded APC into lymph nodes.

Purpose: We have previously reported a clinical trial on the intravenous injection of autologous activated macrophages (AAM) in 15 patients with renal carcinoma. The present paper concerns scintigraphic investigations performed in 11 of these patients after injection of 111indium oxinate-radiolabeled AAM., Methods: AAM were prepared from mononuclear cells (MNC) collected by apheresis from patients treated simultaneously with granulocyte-macrophage colony-stimulating factor (GM-CSF). MNC were cultured for 6 days in the presence of GM-CSF and exposed for 18 h to gamma-interferon, the AAM were then separated by elutriation and injected., Results: After intravenous infusion, radiolabeled AAM were transiently retained in the lungs, where they predominated in the first hour. Later on, radioactivity accumulated in liver and spleen and then decreased from the first and second day, respectively. In one patient, two foci of radioactivity were detected in the lungs 1 h after injection, and persisted thereafter. Their association with tumor lesions was uncertain. This observation possibly resulted from the presence of granulocytes in the radiolabeled AAM populations of this patient. It seems that MNC collected from GM-CSF-treated patients and cultured in the presence of GM-CSF enables the differentiation of granulocytes., Conclusions: A series of 11 investigations confirms the previously reported distribution pattern of intravenously injected AAM. It is possible that in patients treated with hematopoietic cell-mobilizing agents, granulocytes develop in cultures designed to produce monocyte-derived antigen-presenting cells.

Fifteen patients with progressive metastatic renal cell carcinoma were treated with granulocyte-macrophage colony-stimulating factor and intravenous infusions of activated autologous macrophages (AAMs). The latter were prepared from leukapheresis-separated mononuclear cells cultured in the presence of granulocyte-macrophage colony-stimulating factor, exposed to gamma interferon, and submitted to elutriation to separate AAMs. Three intravenous injections of AAMs were performed within a 2-week interval. This treatment cycle was repeated once or twice, in cases of tumor response or stabilization. Ninety-seven preparations containing a mean 3 x 10(9) AAMs were administered and usually well tolerated. One partial response, eight stabilizations and six progressions were observed. The median time to progression and median overall survival time after inclusion were 7 and 9 months, respectively. The cells injected did not accumulate substantially in tumor lesions, as shown by scintigraphic imaging of indium-111-labeled AAMs. Thus, combined granulocyte-macrophage colony-stimulating factor and AAM treatment was well tolerated and resulted in transitory stabilization (n = 8) or partial regression (n = 1) in 9 of 15 patients.

In this study we report the supportive activity of rat liver epithelial cells (RLEC) on human haemopoiesis in the absence of exogeneously supplied growth factors. RLEC is a rat cell line derived from primitive biliary cells with epithelial characteristics which induce the long-term differentiation of hepatocytes through cell-cell contacts. We have established the ability of these cells to sustain long-term survival and multilineage differentiation of human haemopoietic progenitors from unfractionated bone marrow and growth-factor mobilized peripheral blood cells, and from human CD34+ and CD34+ CD38- haemopoietic cells, with a higher efficiency than the murine MS-5 stromal cell line: the numbers of committed progenitors recovered from RLEC cocultures after 8 weeks were 3-fold higher than from MS-5 cocultures, with an unusually high BFU-E production. Furthermore, using diffusible insert cultures, we demonstrated that, despite the lack of strong adhesive interaction between haemopoietic cells and RLEC, physical proximity was absolutely required for optimum stimulation of LTC-IC by RLEC. Taken together, these results show that biliary epithelial cells support human haemopoiesis and cause speculation that common mechanisms might be used by RLEC to regulate both the hepatocyte and the haemopoietic progenitors differentiation.

Unrelated donor searches for 100 Caucasian patients were referred to France Greffe de Moëlle Registry (FGM) from September 1987 (24,600 donors) to December 1993 (71,500 donors, 61% DR typed). After DR typing of HLA-A,B matched donors, unsuccessful searches were extended to other European Registries for 36 patients. Twenty two patients had a donor (FGM: 19, other Registries: 3) selected on: (1) HLA-A,B and DRB,DQB1 split identity; and (2) unidirectional relative response < 5% in MLR performed twice. Estimated probability of finding a compatible donor at 9 months in FGM was 12% (s.e. +/- 4%) and 25% at 2 years (s.e. +/- 6%). This probability was stringently dependent on a phenoidentity to one very common HLA-A,B,DR or B,DR haplotype (25% at 9 months when present, representing 19 of 19 patients with a compatible donor). Without this phenoidentity, the probability was zero per cent (P = 0.0001) in FGM searches and < 4% (n = 1) in extended searches. The MLR test was shown to be insensitive for screening for DPB1 mismatches. Clinical status influenced the probability of finding a compatible donor at one year ranging from 9% +/- 9% for ALL to 23% +/- 8% for CML (NS). Disregarding DPB1 mismatches is the most efficient way of increasing search efficiency.