An accelerated, clinical-grade protocol to generate high yields of type 1-polarizing messenger RNA-loaded dendritic cells for cancer vaccination.

BACKGROUND: Many efforts have been devoted to improve the performance of dendritic cell (DC)-based cancer vaccines. Ideally, a DC vaccine should induce robust type 1-polarized T-cell responses and efficiently expand antigen (Ag)-specific cytotoxic T-cells, while being applicable regardless of patient human leukocyte antigen (HLA) type. Production time should be short, while maximally being good manufacturing practice (GMP)-compliant. We developed a method that caters to all of these demands and demonstrated the superiority of the resulting product compared with DCs generated using a well-established "classical" protocol. METHODS: Immunomagnetically purified monocytes were cultured in a closed system for 3 days in GMP-compliant serum-free medium and cytokines, and matured for 24 h using monophosphoryl lipid A (MPLA)+ interferon-gamma (IFN-γ). Mature DCs were electroporated with messenger RNA (mRNA) encoding full-length antigen and cryopreserved. "Classical" DCs were cultured for 8 days in flasks, with one round of medium and cytokine supplementation, and matured with tumor necrosis factor alpha (TNF-α) + prostaglandin E2 (PGE2) during the last 2 days. RESULTS: Four-day MPLA/IFN-γ-matured DCs were superior to 8-day TNF-α/PGE2-matured DCs in terms of yield, co-stimulatory/co-inhibitory molecule expression, resilience to electroporation and cryopreservation and type 1-polarizing cytokine and chemokine release after cell thawing. Electroporated and cryopreserved DCs according to our protocol efficiently present epitopes from tumor antigen-encoding mRNA, inducing a strong expansion of antigen-specific CD8+ T-cells with full cytolytic capacity. CONCLUSION: We demonstrate using a GMP-compliant culture protocol the feasibility of generating high yields of mature DCs in a short time, with a superior immunogenic profile compared with 8-day TNF-α/PGE2-matured DCs, and capable of inducing vigorous cytotoxic T-cell responses to antigen from electroporated mRNA. This method is now being applied in our clinical trial program.

Radiation Sensitivity of Human CD34(+) Cells Versus Peripheral Blood T Lymphocytes of Newborns and Adults: DNA Repair and Mutagenic Effects.

As hematopoietic stem and progenitor cells (HSPCs) self-renew throughout life, accumulation of genomic alterations can potentially give rise to radiation carcinogenesis. In this study we examined DNA double-strand break (DSB) induction and repair as well as mutagenic effects of ionizing radiation in CD34(+) cells and T lymphocytes from the umbilical cord of newborns. The age dependence of DNA damage repair end points was investigated by comparing newborn T lymphocytes with adult peripheral blood T lymphocytes. As umbilical cord blood (UCB) contains T lymphocytes that are practically all phenotypically immature, we examined the radiation response of separated naive (CD45RA(+)) and memory (CD45RO(+)) T lymphocytes. The number of DNA DSBs was assessed by microscopic scoring of γ-H2AX/53BP1 foci 0.5 h after low-dose radiation exposure, while DNA repair was studied by scoring the number of residual γ-H2AX/53BP1 foci 24 h after exposure. Mutagenic effects were studied by the cytokinesis block micronucleus (CBMN) assay. No significant differences in the number of DNA DSBs induced by low-dose (100-200 mGy) radiation were observed among the three different cell types. However, residual γ-H2AX/53BP1 foci levels 24 h postirradiation were significantly lower in CD34(+) cells compared to newborn T lymphocytes, while newborn T lymphocytes showed significantly higher foci yields than adult T lymphocytes. No significant differences in the level of radiation-induced micronuclei at 2 Gy were observed between CD34(+) cells and newborn T lymphocytes. However, newborn T lymphocytes showed a significantly higher number of micronuclei compared to adult T lymphocytes. These results confirm that CD34(+) cell quiescence promotes mutagenesis after exposure. Furthermore, we can conclude that newborn peripheral T lymphocytes are significantly more radiosensitive than adult peripheral T lymphocytes. Using the results from the comparative study of radiation-induced DNA damage repair end points in naive (CD45RA(+)) and memory (CD45RO(+)) T lymphocytes, we could demonstrate that the observed differences between newborn and adult T lymphocytes can be explained by the immunophenotypic change of T lymphocytes with age, which is presumably linked with the remodeling of the closed chromatin structure of naive T lymphocytes.

In vitro generation of mature, naive antigen-specific CD8(+) T cells with a single T-cell receptor by agonist selection.

Peripheral blood T cells transduced with a tumor-specific T-cell receptor (TCR) face problems of auto-reactivity and lack of efficacy caused by cross-pairing of exogenous and endogenous TCR chains, as well as short term in vivo survival due to activation and growth factor-induced differentiation. We here studied an alternative strategy for the efficient generation of naive CD8(+) T cells with a single TCR. TCR-transduced human postnatal thymus-derived and adult mobilized blood-derived hematopoietic progenitor cells (HPCs) were differentiated to CD4(+)CD8(+) double-positive T cells using OP9-Delta-like 1 (OP9-DL1) cultures. Addition of the agonist peptide induced double positive cells to cross-present the peptide, leading, in the absence of co-stimulation, to cell cycle arrest and differentiation into mature CD8(+) T cells. Comprehensive phenotypic, molecular and functional analysis revealed the generation of naive and resting CD8(+) T cells through a process similar to thymic positive selection. These mature T cells show a near complete inhibition of endogenous TCRA and TCRB rearrangements and express high levels of the introduced multimer-reactive TCR. Upon activation, specific cytokine production and efficient killing of tumor cells were induced. Using this strategy, large numbers of high-avidity tumor-specific naive T cells can be generated from readily available HPCs without TCR chain cross-pairing.

Recommendations in the event of a suspected transfusion-related acute lung injury (TRALI).

The following recommendations, which aim at improving the clinical diagnosis ofTRALI and the laboratory investigations that can support it, were drawn up by a working group of the Superior Health Council. TRALI is a complication of blood transfusion that is both serious and underreported. Systematic reporting may help to develop preventive actions. Therefore, the Superior Health Council recommends that there should be a more stringent surveillance of patients who receive a blood component transfusion. The clinician should pay very close attention to any change in the patient's respiratory status (cf. dyspnoea and arterial desaturation), which should be notified systematically to the haemovigilance contact person in the hospital.

Notch induces human T-cell receptor γδ+ thymocytes to differentiate along a parallel, highly proliferative and bipotent CD4 CD8 double-positive pathway.

In wild-type mice, T-cell receptor (TCR) γδ(+) cells differentiate along a CD4 CD8 double-negative (DN) pathway whereas TCRαß(+) cells differentiate along the double-positive (DP) pathway. In the human postnatal thymus (PNT), DN, DP and single-positive (SP) TCRγδ(+) populations are present. Here, the precursor-progeny relationship of the various PNT TCRγδ(+) populations was studied and the role of the DP TCRγδ(+) population during T-cell differentiation was elucidated. We demonstrate that human TCRγδ(+) cells differentiate along two pathways downstream from an immature CD1(+) DN TCRγδ(+) precursor: a Notch-independent DN pathway generating mature DN and CD8αα SP TCRγδ(+) cells, and a Notch-dependent, highly proliferative DP pathway generating immature CD4 SP and subsequently DP TCRγδ(+) populations. DP TCRγδ(+) cells are actively rearranging the TCRα locus, and differentiate to TCR(-) DP cells, to CD8αß SP TCRγδ(+) cells and to TCRαß(+) cells. Finally, we show that the γδ subset of T-cell acute lymphoblastic leukemias (T-ALL) consists mainly of CD4 SP or DP phenotypes carrying significantly more activating Notch mutations than DN T-ALL. The latter suggests that activating Notch mutations in TCRγδ(+) thymocytes induce proliferation and differentiation along the DP pathway in vivo.

Human intrathymic development: a selective approach.

Human T lymphocytes can be generated from CD34 progenitor cells from different sources. This can be obtained in an in vivo model wherein human thymic tissue and fetal liver is transplanted in an immunodeficient mouse. However, human T cells are also generated in immunodeficient mice without co-transplantation of human thymus or in in vitro hybrid human-mouse fetal thymus organ culture. This shows that xenogeneic mouse thymus tissue supports human T cell differentiation. Finally, human T cells are generated on co-culture with murine stromal cells that express the Delta-like1 ligand for the Notch receptor. How these different environments influence the human T cell repertoire is reviewed and discussed.

Multicentre, prospective, open study to evaluate the safety and efficacy of hylan G-F 20 in knee osteoarthritis subjects presenting with pain following arthroscopic meniscectomy.

The aim of the study was to evaluate the safety and efficacy of viscosupplementation with hylan G-F 20 in patients with mild to moderate osteoarthritis (OA) presenting with persistent knee pain 4-12 weeks after arthroscopic meniscectomy. A prospective, multi-centre, open study was carried out in patients with pain due to OA of the knee, not resolved by simple analgesics, 4-12 weeks after undergoing arthroscopic meniscectomy. To be eligible, patients had to score > or =50 mm and < or =90 mm on both walking pain and patient global assessment visual analogue scales (VAS; 0-100 mm) at baseline and be radiologically diagnosed pre-operatively with OA grade I or II on the Kellgren-Lawrence scale, with <50% joint space narrowing. Patients received three intra-articular, 2 ml injections of hylan G-F 20 in the target knee with an interval of 1 week between injections, and were followed for 52 weeks. The primary efficacy endpoint was the change from baseline in the walking pain VAS score at 26 weeks. Secondary outcome measures were the walking pain VAS scores at all other time points, the WOMAC Index at all time points, and patient and physician global assessment at all time points. The safety of the treatment was assessed using adverse event (AE) reports. A total of 62 patients (mean age 55.4 years, 52% male) were enrolled. The mean walking pain VAS score decreased by 36.8 mm from baseline at 26 weeks (P < 0.0001), and also showed statistically significant decreases (P < 0.0001) at all other time points. The change in WOMAC total and subscale scores from baseline were statistically significant (P < 0.0001) at all time points, as were the decreases in the physician and patient global assessment VAS scores. There were 18 target knee AEs (mostly pain and/or swelling and/or effusion) in 12 patients (19%) considered to be at least possibly related to treatment. The majority of these (78%) were mild or moderate in intensity. One patient (1.6%) experienced a serious adverse event (synovitis) in the target knee that was considered possibly related to study treatment. Hylan G-F 20 provides effective pain relief and improves stiffness and physical function in patients with mild to moderate OA presenting with persistent osteoarthritic pain 4-12 weeks after arthroscopic meniscectomy. Symptomatic efficacy was maximised at 12 weeks and maintained at 26 and 52 weeks. The type (pain and/or swelling and/or effusion) and the intensity (mostly mild/moderate) of AEs reported in this study are similar to those reported in other trials in different patient populations, but the incidence was higher (19%). The risk/benefit of hylan G-F 20 in this particular population of patients is favourable.

Guidelines for the use of fresh frozen plasma.

Recommendations, which aim at standardising and rationalising clinical indications for the transfusion of fresh frozen plasma (FFP) in Belgium, were drawn up by a working group of the Superior Health Council. For this purpose the Superior Health Council organised an expert meeting devoted to "Transfusion Guidelines: Pathogen reduction, products and indications for the transfusion of plasma" in collaboration with the Belgian Haematological Society.The experts discussed the indications for the transfusion of FFP, pathogen reduction for FFP and the practical issues of administering FFP and plasma-derived concentrates. The recommendations formulated by the experts were validated by the working group with the purpose of harmonising FFP transfusion in Belgian hospitals.

The safety and efficacy of a powered toothbrush on soft tissues in patients with implant-supported fixed prostheses.

The safety, efficacy and acceptability of an oscillating/rotating powered toothbrush was assessed in patients rehabilitated with fixed prostheses on implants. One hundred consecutive patients (aged 18-80; mean 56.3; 51 females), who met the inclusion/exclusion criteria and who participated in a regular annual recall scheme, were enrolled. They were instructed on how to use the powered toothbrush, as well as on classical interdental plaque control. The electric toothbrush had to be used twice daily for 2 min. The following periodontal parameters were measured at baseline and at 3 months, 6 months and 12 months: presence/absence of gingival and/or mucosal ulceration/desquamation; sulcus bleeding index; probing pocket depth; periodontal pocket-bleeding index and gingival recession. At 3 months and at the end of the study, patients completed a questionnaire concerning the overall acceptability and convenience of the powered toothbrush, as compared with their habitual manual toothbrush. A total of 80 patients completed the study. No dropouts were related to the use of the powered toothbrush. All parameters improved over the course of the study. The mean overall pocket depth decreased from 3.3 mm at baseline to 3.0 mm at 12 months, while the mean decrease in recession was 0.1 mm at 12 months. During the 1-year observation, there was a slight gain in periodontal attachment level. Gingival ulcerations were not observed at any point in the study. High scores for convenience and comfort of the powered toothbrush were reported, and the majority (95%) said that they would continue to use it for habitual oral hygiene. It is concluded that the powered toothbrush investigated is effective, safe and comfortable for patients rehabilitated by means of oral implant-supported prostheses.

The immediate rehabilitation by means of a ready-made final fixed prosthesis in the edentulous mandible: a 1-year follow-up study on 50 consecutive patients.

Although a two-stage procedure with symphyseal oral implants can lead to a long-term (15 years) 99% cumulative survival rate, a one-stage approach with immediate loading via prefabricated elements seems to offer a short-term acceptable outcome with significantly lower costs. A series of 50 consecutive patients, not eliminated for any systemic or smoking condition, received at the department of periodontology three implants in the symphyseal area, connected by a very rigid horse shoe-shaped titanium bar. A final screw retained prosthetic framework was placed on top of it at the department of prosthetic dentistry within 2 days after surgery (44) or after a delay, due to purely external factors, of up to 10 days (6). Forty-five patients were followed for 1 year. In one patient, all three implants failed and another four patients were lost to follow-up. The cumulative failure rates for implants and prostheses at 1 year were, respectively, 7.3% and 5%. The mean marginal bone loss at 1 year was 1.08 mm (SD: 1.62; range -5.68 to +2.55). This study shows that stable marginal bone levels can be maintained around immediately loaded implants in the lower jaw in an average patient population for at least 1 year. The survival rate is, however, lower than for a staged approach.

[The role of periodontal diseases in bad breath]. / De rol van parodontale aandoeningen bij ademgeurklachten.

The majority of bad breath originates within the oral cavity. However, it is also possible that it is caused by other extra-oral sources. Ear-nose-and-throat causes and systemic diseases, such as diabetes mellitus, uremia, liver failure, pulmonary carcinoma, bronchiectasis, are the most frequent problems. In 65-85% of the cases of bad breath, we find the cause in the parodontium and/or tongue. Components from tongue coating and periodontal pockets circulate through the saliva in the whole oral cavity and also reach the tonsils.

Arthroscopic partial lateral meniscectomy long-term results in athletes.

This is a retrospective case-control study of arthroscopic partial meniscectomy for isolated lesions of the lateral meniscus, performed between 1990 and 1995. Thirty-one knees were evaluated after an average follow-up of 8 years: 48.4% had excellent/good IKDC-scores and 64.5% excellent/good Lysholm scores. The Tegner activity score dropped from 7.2 (competitive sports) to 5.7 (recreational sports). Fairbank changes were noted in 92.9% of the radiographs. Deterioration of results after arthroscopic partial lateral meniscectomy is obvious. The extent of the resection is a significant factor.

Both CD34+38+ and CD34+38- cells home specifically to the bone marrow of NOD/LtSZ scid/scid mice but show different kinetics in expansion.

Human hemopoietic stem cells (HSC) have been shown to engraft, differentiate, and proliferate in the hemopoietic tissues of sublethally irradiated NOD/LtSZ scid/scid (NOD/SCID) mice. We used this model to study homing, survival, and expansion of human HSC populations from different sources or phenotype. We observed that CD34+ cells homed specifically to bone marrow (BM) and spleen, but by 3 days after injection, survived only in the BM. These BM-homed CD34+ cells proliferated intensively and gave rise to a 12-fold, 5.5-fold, and 4-fold expansion in 3 days for umbilical cord blood, adult mobilized peripheral blood, and adult BM-derived cells, respectively. By injection of purified subpopulations, it was demonstrated that both CD34+38+ and CD34+38- umbilical cord blood HSC homed to the BM and expanded. Importantly, kinetics of expansion were different: CD34+38+ cells started to increase in cell number from day 3 onwards, and by 4 wk after injection, virtually all CD34+ cells had disappeared. In contrast, CD34+38- cells remained quiescent during the first week and started to expand intensively from the third week on. In this paper, we have shown that homing, survival, and expansion of stem cells are three independent phenomena important in the early phase of BM engraftment and that kinetics of engraftment differ between CD34+38+ and CD34+38- cells.

Human T lymphopoiesis. In vitro and in vivo study models.

Successive steps in T lymphocyte differentiation and T potential of human stem cells (HSC) can be tested in the following models: (a) the infusion of cells in NOD-SCID mice, (b) the injection of cells in renconstituted SCID/hu mice, (c) the differentiation of cells in fetal thymus organ culture (FTOC), and (d) on thymic stromal layers. Using mixed human-murine FTOC, we showed (a) TCR alpha beta, TCR gamma delta lymphocytes, NK cells, and dendritic cells complete their differentiation, (b) IL-7R alpha signaling and IL-7 are essential, (c) a detailed phenotypic and functional analysis of discrete successive steps of positively selected thymocytes, (d) an efficient transduction of genes in HSC with persistent gene expression throughout the T-lymphocyte differentiation, and (e) adaptation to submerging high oxygen culture increases the test sensitivity to a clonal assay. Other approaches are the in vivo SCID/hu reconstitution model. With this method small fragments of human fetal liver and thymus are implanted under the kidney capsule of an adult SCID mouse with result in an impressive human thymus organ, six months after transplantation. We use this model to study thymus T-cell developmental kinetics, development of gene-marked precursor cells and thymic homing of precursor cells.

Thymic repopulation by CD34(+) human cord blood cells after expansion in stroma-free culture.

Thymic repopulation by transplanted hematopoietic progenitor cells (HPC) is likely to be important for long-term immune reconstitution and for successful gene therapy of diseases affecting the T-cell lineage. However, the T-cell progenitor potential of HPC, cultured in vitro for cell number expansion and gene transfer remains largely unknown. Here, we cultured highly purified human umbilical cord blood (CB) CD34(+)CD38(-) or CD34(+)CD38(+) cells for up to 5 weeks in stroma-free cultures supplemented with various combinations of the cytokines thrombopoietin (TPO), stem cell factor (SCF), flt3/flk-2 ligand (FL), interleukin-3 (IL-3), and IL-6 and investigated thymus-repopulating ability of expanded cells in vitro and in vivo. After up to 5 weeks of culture in IL-3 + SCF + IL-6 or TPO + FL + SCF supplemented medium, the progeny of CD34(+)CD38(-) CB cells generated T cells and natural killer cells in the thymus. Limiting dilution experiments demonstrated increase in the number of T-cell progenitors during culture. After 3 weeks of culture, gene marked CD34(+)CD38(-) CB cells injected in the human thymus fragment transplanted in severe combined immunodeficient (SCID) mice (SCID-hu) generated thymocytes expressing the retroviral encoded marker gene GFP in vivo. Thus, our results show that the progeny of CD34(+)CD38(-) CB cells cultured for extensive periods, harbor thymus-repopulating cells that retain T-cell progenitor potential after expansion and gene transfer.

The analgesic effect of preoperative administration of propacetamol, tenoxicam or a mixture of both in arthroscopic, outpatient knee surgery.

A prospective, randomized, double-blind, placebo-controlled, comparative study was undertaken to assess the efficacy of the preemptive use of propacetamol, tenoxicam or the combination of both in arthroscopic, outpatient surgery of the knee. One hundred patients aged 18 to 65 years, ASA 1-2, scheduled for arthroscopy were randomized to receive propacetamol 30 mg/kg i.v. (repeated after 6 hours), tenoxicam 0.5 mg/kg i.v. (max. 40 mg), the combination of both or placebo one hour prior to a standard anesthetic. There were no differences with regard to total dose opioid consumption, sedation scores and side effects in the four groups.

In vitro intrathymic differentiation kinetics of human fetal liver CD34+CD38- progenitors reveals a phenotypically defined dendritic/T-NK precursor split.

Human CD34+CD38- hematopoietic precursor cells from fetal liver are able to develop into T, NK, and dendritic cells in a hybrid human/mouse fetal thymic organ culture (FTOC). In this report, we pay particular attention to the early events in differentiation of these precursor cells. We show that the CD34+CD38- precursor cells, which are CD4-CD7-cyCD3-HLA-DR-/++ (cy, cytoplasmatic), differentiate into a CD4+ population that remained CD7-cyCD3-HLA-DR++ and a CD4- population that expressed CD7 and cyCD3. The CD4+CD7-cyCD3- cells differentiate into phenotypically and functionally mature dendritic cells, but do not differentiate into T or NK cells. The CD4-CD7+cyCD3+ population later differentiates into a CD4+CD7+cyCD3+HLA-DR- population, which has no potential to differentiate into dendritic cells but is able to differentiate into NK cells and gammadelta and alphabeta T lymphocytes. These findings support the notion that the T/NK split occurs downstream of the NK/dendritic split.

An inventory of patients' response to treatment at a multidisciplinary breath odor clinic.

OBJECTIVE: This study was undertaken to assess patients' response to their treatment at a multidisciplinary oral malodor clinic. METHOD AND MATERIALS: In 4 years, a multidisciplinary breath odor clinic in Belgium examined 406 patients. The team consisted of an ear, nose, and throat specialist, a periodontologist, occasionally a specialist in internal medicine, and, more recently, a psychiatrist. After the initial visit, each patient was scheduled for a follow-up appointment 2 to 6 months later; however, only 143 patients (35%) showed up for this control visit. The remaining 65% of the patients answered a mailed questionnaire. RESULTS: About half of the patients who returned no longer had complaints, while 17% reported no improvement. This group was characterized by imaginary bad breath and manifest psychologic problems. There was also disbelief of their cure, although clinical examination (organoleptic evaluation and volatile sulfide measurement by means of a portable monitor) did not reveal any oral malodor. Some also insufficiently performed the recommended oral hygiene measures (tongue brushing and interdental cleaning). Most of the patients who returned the questionnaire were disappointed by the suggestion that their halitosis was the result of insufficient oral hygiene. CONCLUSION: Better education of both the public and dental professionals as to the most frequent cause of halitosis, insufficient oral hygiene, might elevate the level of compliance by patients.

The use of locally delivered minocycline in the treatment of chronic periodontitis. A review of the literature.

Tetracyclines are frequently used in the treatment of periodontitis; however, emergence of resistant bacterial strains has decreased the utility of these drugs. At present, there are a lot of data in the literature from which one can draw conclusions regarding the use of local drug delivery. This paper reviews the utility and different systems of local delivery of minocycline, a semisynthetic tetracycline, in the treatment of periodontitis.